Methods of determining susceptibility to or presence of schizophrenia, or a disorder related thereto

ABSTRACT

Variations in the DNA sequence of the human proline dehydrogenase gene (PRODH) which correlate to an increased susceptibility to, or presence of schizophrenia or a disease or disorder related thereto, such as obsessive compulsive disorder (OCD), bipolar disorder (BP), or major depressive disorder (MDD) are provided, along with assays to diagnosing schizophrenia or a disease or disorder related thereto, and evaluating potential drugs or agents for using in treating such diseases or disorders.

CROSS REFERENCE TO A RELATED APPLICATION

[0001] This Application is a continuation-in-part of copending U.S.application Ser. No. 09/229,530 entitled “Methods of DeterminingSusceptibility to or presence of schizophrenia, or a disorder relatedthereto”, which is hereby incorporated by reference in its entirety.

FIELD OF THE INVENTION

[0002] The present invention relates to isolated nucleic acid moleculeswhich encode human and murine proline dehydrogenase, and methods fordetermining susceptibility to, or the presence of schizophrenia or adisease or disorder related thereto, such as obsessive compulsivedisorder, bipolar disorder (BP), or major depressive disorder in asubject by determining levels of proline dehydrogenase (PRODH) in abodily sample. Furthermore, the present invention comprisespolymorphisms of the human proline dehydrogenase (PRODH) gene whichcorrelate to a phenotype closely related to schizophrenia or a diseaseor disorder related thereto. The present invention also relates tovarious assays for drugs or agents that can treat schizophrenia or adisease or disorder related thereto.

BACKGROUND OF THE INVENTION

[0003] It has been posited that the amino acid proline serves as amodulator of synaptic transmission in the mammalian brain, due to theselective expression of a brain specific high affinity prolinetransporter in a subset of glutamatergic pathways (Fremeau et al.,1996). Proline transporter is modulated by enckephalins, the expressionof which may be decreased in the brains of patients with schizophreniaand elevated proline concentration. Furthermore, recent analysisindicates that endogenous extracellular proline may regulate the basalfunction of some glutamate synapses by maintaining them in a partiallypotentiated state. Also, elevated proline concentration has also beenpreviously associated with behavioral and neurological effects.

[0004] Evidence of an association between schizophrenia susceptibilityand hemizygous deletions in chromosome 22q11 has been reported. Morespecifically, three hemizygous cryptic deletions at 22q11 in a sample of300 unrelated schizophrenic patients have been reported andcharacterized [Karayiorgou et al., Proc. Natl. Acad. Sci. U.S.A. 92,7612 (1995); Karayiorgou et al., Amer. J. Med. Genet. 74, 677 (1997)].The frequency of this micro-deletion in the general population isestimated to be approximately 0.02% and no deletions were found in asample of 200 healthy controls. The identified locus (approximately 1.5Mb in size) is located in the proximal part of a region at chromosome22q11 and has been implicated independently in schizophreniasusceptibility through linkage studies [Karayiorgou & Gogos, Neuron 19,967-979 (1997)]. This locus overlaps with the critical region involvedin the etiology of Velocardio-facial (VCFS)/DiGeorge (DGS) syndromes[Driscoll et al. 1993]. Furthermore, it has been shown thatapproximately 29% VCFS children with 22q11 deletions developschizophrenia or schizoaffective disorder in adolescence and adulthood[Pulver et al., 1994], an estimate confirmed by a more recentindependent study [Murphy and Owen, Am. J. Med. Genet., 74, 660 (1997)].Deletions in chromosome 22, band q11 (22q11) have been identified amongschizophrenia patients of diverse ethnic origins (Chinese, Israeli,British, Danish [L. Y. Chow et al., Am. J. Med. Genet. 74, 677 (1997);D. Gothelf et al., Am. J. Med. Genet. 72, 455 (1997); 0. Mors and H.Ewald, Am. J. Med. Genet. 74, 677 (1997); Hodginson et al, Am. J. Med.Genet. 61, 565 (1997)]) and the 22q11 region has been implicated inearly-onset schizophrenia [Yan et al., 1998]. In addition, the increasedrates of comorbid obsessive compulsive disorder (OCD) or symptoms (OCS)among schizophrenic patients with the 22q11 microdeletion locus[Karayiorgou et al., 1996, 1997; Papolos et al., 1996] and similarlyincreased rates of anxiety, OCS and OCD in children and adults with the22q11 microdeletion in the absence of schizophrenia [Papolos et al.,1996], potentially indicate that the 22q11 genomic region may harbor oneor more genes predisposing to obsessive compulsive disorder (OCD).

[0005] Moreover, it has been observed that approximately 20% ofschizophrenia patients report obsessions and compulsions, features thatare found in only 1-2% of the general population [Eisen & Rasmussen1993; Berman et al., 1995]. Hence, it is possible that schizophrenia andOCD may share some pathophysiological and genetic components. One commoncentral processing mechanism that seems to be affected in patients withschizophrenia and OCD is sensorimotor gating. Patients withschizophrenia and OCD demonstrate poor sensorimotor gating of thestartle response as measured by impaired prepulse inhibition of anacoustic response, and this may lead to sensory overload,distractibility and cognitive fragmentation.

[0006] However, there is no genetic marker available which is indicativeof a subject's susceptibility to schizophrenia, or a disease relatedthereto, such as obsessive compulsive disorder (OCD), bipolar disorder(BP), or major depressive disorder (MDD).

[0007] Accordingly, what is needed is a genetic marker to assess asubject's susceptibility to schizophrenia or a disease or disorderrelated thereto. Also needed is a genetic marker to diagnoseschizophrenia, and the development of potential drugs or agents thathave applications in treating schizophrenia or a disease or disorderrelated thereto, such as OCD, bipolar disorder, or major depressivedisorder.

[0008] The citation of any reference herein should not be construed asan admission that such reference is available as “Prior Art” to theinstant application.

SUMMARY OF THE INVENTION

[0009] There is provided, in accordance with the present invention, anisolated nucleic acid molecule which encodes human prolinedehydrogenase, and the amino acid sequence of human prolinedehydrogenase. Also provided is an isolated nucleic acid moleculecomprising a DNA sequence which encodes murine proline dehydrogenase,and the amino acid sequence of murine proline dehydrogenase.Furthermore, there is provided, in accordance with the presentinvention, methods for determining a subject's susceptibility toschizophrenia or a disease or disorder related thereto, such as aschizoaffective disorder or disorders related thereto, like OCD, bipolardisorder, or major depressive disorder, using a variant allele of thegene encoding PRODH. Detection of such a variant allele in the genome ofa subject may be indicative of the subject's susceptibility toschizophrenia. Furthermore, a variant allele of the PRODH gene can alsobe used to assay drugs and agents for potential use in treatingschizophrenia or a disease or disorder related thereto, such asobsessive compulsive disorder (OCD), bipolar disorder (BP) or majordepressive disorder (MDD).

[0010] Thus broadly, the present invention extends to an isolatednucleic acid molecule encoding human proline dehydrogenase, wherein theisolated nucleic acid molecule comprises a DNA sequence of SEQ ID NO:1,degenerate variants thereof, fragments thereof, or analogs orderivatives thereof.

[0011] Furthermore, the present invention extends to an isolated nucleicacid molecule hybridizable under standard hybridization conditions tothe isolated nucleic acid molecule comprising a DNA sequence of SEQ IDNO:1, degenerate variants thereof, fragments thereof, or analogs orderivatives thereof.

[0012] The present invention also extends to an isolated nucleic acidmolecule comprising a DNA sequence of SEQ ID NO:1, degenerate variantsthereof, fragments thereof, or analogs or derivatives thereof, or anisolated nucleic acid molecule hybridzable thereto under standardhybridization conditions, wherein the nucleic acid molecule isdetectably labeled. Numerous detectable labels have applications in thepresent invention. Examples include a radioactive element, such as theisotopes ³H, ¹⁴C, ³²P, ³⁵S, ³⁶Cl, ⁵¹Cr, ⁵⁷Co, ⁵⁸Co, ⁵⁹Fe, ⁹⁰Y, ²⁵¹I,¹³¹I, and ¹⁸⁶Re, to name only a few, chemicals which fluoresce, orenzymes such as alkaline phosphatase or horseradish peroxidaseconjugated to an isolated nucleic acid molecule of the invention.

[0013] Moreover, the present invention extends to an isolated nucleicacid molecule comprising a DNA sequence of SEQ ID NO:1, degeneratevariants thereof, fragments thereof, or analogs or derivatives thereof,or an isolated nucleic acid molecule hybridizable under standardhybridization conditions thereto, wherein the nucleic acid moleculeencodes human PRODH comprising an amino acid sequence of SEQ ID NO:2,conservative variants thereof, fragments thereof, or analogs orderivatives thereof.

[0014] Naturally, the present invention extends to an isolated humanproline dehydrogenase protein (PRODH) comprising an amino acid sequenceof SEQ ID NO:2, conservative variants thereof, fragments thereof, oranalogs or derivatives thereof.

[0015] Also, the present invention extends to an antibody having humanproline dehydrogenase comprising an amino acid sequence of SEQ ID NO:2,conservative variants thereof, fragments thereof, or analogs orderivatives thereof, as an immunogen. Such an antibody can bepolyclonal, monoclonal, or chimeric. Further, an antibody of theinvention having human PRODH as an immunogen can be detectably labeled.As explained above, examples of detectable labels having applicationsherein include, but certainly are not limited to radioactive isotopes,such as ³H, ¹⁴C, ³²P, ³⁵S, ³⁶Cl, ⁵¹Cr, ⁵⁷Co, ⁵⁸Co, ⁵⁹Fe, ⁹⁰Y, ¹²⁵I,¹³¹I, and ¹⁸⁶Re, to name only a few. Chemicals which fluoresce, orenzymes such as alkaline phosphatase or horseradish peroxidase, can alsobe used as detectable labels.

[0016] In addition, the present invention extends to cloning vectors forcreating copies or “cloning” an isolated nucleic acid molecule of theinvention. More specifically, the present invention extends to a cloningvector comprising an isolated nucleic acid molecule comprising a DNAsequence of SEQ ID NO:1, degenerate variants thereof, fragments thereof,or analogs or derivatives thereof, and an origin of replication. Inanother embodiment, the invention extends to a cloning vector comprisingan origin of replication and an isolated nucleic acid moleculehybridizable under standard hybridization conditions to an isolatednucleic acid molecule comprising a DNA sequence of SEQ ID NO:1,degenerate variants thereof, fragments thereof, or analogs orderivatives thereof.

[0017] Numerous cloning vectors which are commercially available to theskilled artisan can be used as a cloning vector of the invention.Further, it is readily within the skill of one of ordinary skill in theart to insert an isolated nucleic acid molecule comprising a DNAsequence of SEQ ID NO:1, a degenerate variant thereof, a fragmentthereof, or an analog or derivative thereof, or an isolated nucleic acidmolecule hybridizable thereto under standard hybridization conditionsinto a readily available cloning vector using recombinant DNA techniquesto produce a cloning vector of the invention. In particular, numerouscommercially available cloning vectors have a polylinker site. One ofordinary skill in the art can readily cut open a cloning vector at itspolylinker using a variety restriction endonucleases, and then insertthe isolated nucleic acid molecule into the vector using DNA ligase.Furthermore, a skilled artisan can also manipulate the ends of anisolated nucleic acid molecule of the invention or fragment thereof tocomprise particular restriction sites, and cut those cites withrestriction endonucleases which also were used to cut open the vector.The restricted isolated nucleic acid molecule of the invention can thenbe readily inserted into a cloning vector of the invention. Anyremaining gaps in the DNA sequence of the vector can then be filled inusing individual deoxynucleotides and DNA ligase. Particular cloningvectors which have applications in the present invention include, butare not limited to E. coli, bacteriophages such as lambda derivatives,plasmids such as pBR322 derivatives, and pUC plasmid derivatives such aspGEX vectors, or pmal-c, pFLAG, to name only a few.

[0018] Naturally, the present invention extends to an expression vectorfor expressing an isolated nucleic acid molecule of the invention inorder to produce human PRODH, conservative variants thereof, fragmentsthereof, or analogs or derivatives thereof. In particular, an expressionvector of the invention comprises an isolated nucleic acid moleculecomprising a DNA sequence of SEQ ID NO:1, degenerate variants thereof,fragments thereof, or analogs or derivatives thereof, operativelyassociated with a promoter. In another embodiment, an expression vectorof the invention comprises an isolated nucleic acid molecule operativelyassociated with a promoter, wherein the isolated nucleic acid moleculeis hybridzable under standard hybridization conditions to an isolatednucleic acid molecule comprising a DNA sequence of SEQ ID NO:1,degenerate variants thereof, fragments thereof, or analogs orderivatives thereof.

[0019] Numerous expression vectors can be used to express an isolatednucleic acid molecule comprising a DNA sequence of SEQ ID NO:1,degenerate variants thereof, fragments thereof, or analogs orderivatives thereof, or an isolated nucleic acid molecule hybridizablethereto under standard hybridization conditions. In particular, suchexpression vectors are generally commercially available to the skilledartisan, and like cloning vectors, comprise polylinker sites. As aresult, commercially available expression vectors can be manipulated ina fashion similar to to the manipulation of a cloning vector, which isdescribed above. Hence a skilled artisan can readily insert an isolatednucleic acid molecule of the invention into an expression vector suchthat the isolated nucleic acid molecule is operatively associated with apromoter. Examples of expression vectors having applications herein aredescribed infra.

[0020] Moreover, the present invention extends to a unicellular hosttransformed or transfected with an expression vector comprising anisolated nucleic acid molecule operatively associated with a promoter,wherein the isolated nucleic acid molecule encodes a human prolinedehydrogenase protein comprising an amino acid sequence of SEQ ID NO:2,conservative variants thereof, fragments thereof, or analogs orderivatives thereof. In one embodiment, the present invention extends toa unicellular host transformed or transfected with an expression vectorcomprising an isolated nucleic acid molecule operatively associated witha promoter, wherein the isolated nucleic acid molecule comprises a DNAsequence of SEQ ID NO:1, degenerate variants thereof, fragments thereof,or analogs or derivatives thereof. In another embodiment, the inventionextends to a unicellular host transformed or transfected with anexpression vector comprising an isolated nucleic acid moleculeoperatively associated with a promoter, wherein the isolated nucleicacid molecule comprises a DNA sequence hybridizable under standardhybridization conditions to an isolated nucleic acid molecule comprisinga DNA sequence of SEQ ID NO:1, a degenerate variant thereof, a fragmentthereof, or an analog or derivative thereof. Numerous unicellular hostswhich are readily available to the skilled artisan have applications inthe present invention. Examples include, but certainly are not limitedto, E. coli, Pseudonomas, Bacillus, Strepomyces, yeast, CHO, R1.1, B-W,L-M, COS1, COS7, BSC1, BSC40, BMT10 and Sf9 cells.

[0021] Naturally, the present invention extends to method for producinga PRODH comprising an amino acid sequence of SEQ ID NO:2, conservativevariants thereof, fragments thereof, or analogs or derivatives thereof.In one embodiment, the method comprises the steps of:

[0022] a) culturing a unicellular host transformed or transfected withan expression vector comprising an isolated nucleic acid molecule whichcomprises a DNA sequence of SEQ ID NO:1, a degenerate variant thereof, afragment thereof, or an analog or derivative thereof, operativelyassociated with a promoter, under conditions that provide for expressionof the isolated nucleic acid molecule to produce a protein comprising anamino acid sequence of SEQ ID NO:2, a conservative variant thereof, afragment thereof, or analog or derivative thereof; and

[0023] b) recovering the protein from the unicellular host, the culture,or both.

[0024] In another embodiment, the method comprises the steps of:

[0025] a) culturing a unicellular host transformed or transfected withan expression vector comprising an isolated nucleic acid moleculeoperatively associated with a promoter, wherein the isolated nucleicacid molecule is hybridizable under standard hybridization conditions toan isolated nucleic acid molecule comprising a DNA sequence of SEQ IDNO:1, a degenerate variant thereof, a fragment thereof, or an analog orderivative thereof, operatively associated with a promoter, underconditions that provide for expression of the isolated nucleic acidmolecule, to produce a protein comprising an amino acid sequence of SEQID NO:2, a conservative variant thereof, a fragment thereof, or ananalog or derivative thereof; and

[0026] b) recovering the protein from the unicellular host, the culture,or both.

[0027] In another embodiment, the present invention extends to anisolated nucleic acid molecule which encodes a human PRODH, wherein theisolated nucleic acid molecule comprises a DNA sequence of SEQ ID NO:9,degenerate variants thereof, fragments thereof, or analogs orderivatives thereof.

[0028] Furthermore, the present invention extends to an isolated nucleicacid molecule hybridizable under standard hybridization conditions tothe isolated nucleic acid molecule comprising a DNA sequence of SEQ IDNO:9, degenerate variants thereof, fragments thereof, or analogs orderivatives thereof.

[0029] The present invention also extends to an isolated nucleic acidmolecule comprising a DNA sequence of SEQ ID NO:9, degenerate variantsthereof, fragments thereof, or analogs or derivatives thereof, or anisolated nucleic acid molecule hybridzable thereto under standardhybridization conditions, wherein the nucleic acid molecule isdetectably labeled. Numerous detectable labels have applications in thepresent invention and are described infra.

[0030] Moreover, the present invention extends to an isolated nucleicacid molecule comprising a DNA sequence of SEQ ID NO:9, degeneratevariants thereof, fragments thereof, or analogs or derivatives thereof,or an isolated nucleic acid molecule hybridizable under standardhybridization conditions thereto, which encodes human PRODH comprisingan amino acid sequence of SEQ ID NO:2, conservative variants thereof,fragments thereof, or analogs or derivatives thereof.

[0031] In addition, the present invention extends to cloning vectors forcreating copies or “cloning” an isolated nucleic acid molecule of theinvention. More specifically, the present invention extends to a cloningvector comprising an isolated nucleic acid molecule comprising a DNAsequence of SEQ ID NO:9, degenerate variants thereof, fragments thereof,or analogs or derivatives thereof, and an origin of replication. Inanother embodiment, the invention extends to a cloning vector comprisingan origin of replication and an isolated nucleic acid moleculehybridizable under standard hybridization conditions to an isolatednucleic acid molecule comprising a DNA sequence of SEQ ID NO:9,degenerate variants thereof, fragments thereof, or analogs orderivatives thereof.

[0032] Numerous cloning vectors which are commercially available to theskilled artisan can be used as a cloning vector of the invention.Further, it is readily within the skill of one of ordinary skill in theart to insert an isolated nucleic acid molecule of the present inventionor fragment thereof into a readily available cloning vector usingrecombinant DNA techniques to produce a cloning vector of the invention.What's more numerous cloning vectors having applications herein arereadily available to the skilled artisan, such that with the use ofroutine experimental techniques and the new and useful isolated nucleicacid molecule comprising a DNA sequence of SEQ ID NO:9, one of ordinaryskill can readily produce a cloning vector of the invention. Naturally,routine recombinant DNA techniques describe here can apply to theinsertion of any isolated nucleic acid molecule of the invention in to acloning vector.

[0033] Also, the present invention extends to an expression vector forexpressing an isolated nucleic acid molecule of the invention in orderto produce human PRODH, conservative variants thereof, fragmentsthereof, or analogs or derivatives thereof. In particular, an expressionvector of the invention comprises an isolated nucleic acid moleculecomprising a DNA sequence of SEQ ID NO:9, degenerate variants thereof,fragments thereof, or analogs or derivatives thereof, operativelyassociated with a promoter. In another embodiment, an expression vectorof the invention comprises an isolated nucleic acid molecule operativelyassociated with a promoter, wherein the isolated nucleic acid moleculeis hybridzable under standard hybridization conditions to an isolatednucleic acid molecule comprising a DNA sequence of SEQ ID NO:9,degenerate variants thereof, fragments thereof, or analogs orderivatives thereof.

[0034] Numerous expression vectors can be used to express an isolatednucleic acid molecule comprising a DNA sequence of SEQ ID NO:9,degenerate variants thereof, fragments thereof, or analogs orderivatives thereof, or an isolated nucleic acid molecule hybridizablethereto under standard hybridization conditions. In particular, suchexpression vectors are generally commercially available to the skilledartisan, and like cloning vectors, comprise polylinker sites. As aresult, commercially available expression vectors can be manipulated ina similar fashion in which cloning vectors of the invention aremanipulated. Hence a skilled artisan can readily insert an isolatednucleic acid molecule of the invention into an expression vector suchthat the isolated nucleic acid molecule is operatively associated with apromoter. Examples of expression vectors having applications herein aredescribed infra.

[0035] Moreover, the present invention extends to a unicellular hosttransformed or transfected with an expression vector comprising anisolated nucleic acid molecule operatively associated with a promoter,wherein the isolated nucleic acid molecule encodes a human prolinedehydrogenase protein comprising an amino acid sequence of SEQ ID NO:2,conservative variants thereof, fragments thereof, or analogs orderivatives thereof. In one embodiment, the present invention extends toa unicellular host transformed or transfected with an expression vectorcomprising an isolated nucleic acid molecule operatively associated witha promoter, wherein the isolated nucleic acid molecule comprises a DNAsequence of SEQ ID NO:9, degenerate variants thereof, fragments thereof,or analogs of derivatives thereof. In another embodiment, the inventionextends to a unicellular host transformed or transfected with anexpression vector comprising an isolated nucleic acid moleculeoperatively associated with a promoter, wherein the isolated nucleicacid molecule comprises a DNA sequence hybridizable under standardhybridization conditions to an isolated nucleic acid molecule comprisinga DNA sequence of SEQ ID NO:9, a degenerate variant thereof, a fragmentthereof, or an analog or derivative thereof. Numerous unicellular hostswhich are readily available to the skilled artisan have applications inthe present invention, and examples are described above.

[0036] Naturally, the present invention extends to method for producinga PRODH comprising an amino acid sequence of SEQ ID NO:2, conservativevariants thereof, fragments thereof, or analogs or derivatives thereof.In one embodiment, a method for producing human proline dehydrogenasecomprises the steps of:

[0037] c) culturing a unicellular host of transformed or transfectedwith an expression vector comprising an isolated nucleic acid moleculewhich comprises a DNA sequence of SEQ ID NO:9, a degenerate variantthereof, a fragment thereof, or an analog or derivative thereof,operatively associated with a promoter, under conditions that providefor expression of the isolated nucleic acid molecule to produce aprotein comprising an amino acid sequence of SEQ ID NO:2, a conservativevariant thereof, a fragment thereof, or analog or derivative thereof;and

[0038] d) recovering the protein from the unicellular host, the culture,or both.

[0039] In another embodiment, the method comprises the steps of:

[0040] a) culturing a unicellular host of transformed or transfectedwith an expression vector comprising an isolated nucleic acid moleculehybridizable under standard hybridization conditions to an isolatednucleic acid molecule comprising a DNA sequence of SEQ ID NO:9, adegenerate variant thereof, a fragment thereof, or an analog orderivative thereof, operatively associated with a promoter, underconditions that provide for expression of the isolated nucleic acidmolecule, to produce a protein comprising an amino acid sequence of SEQID NO:2, a conservative variant thereof, a fragment thereof, or ananalog or derivative thereof; and

[0041] b) recovering the protein from the unicellular host, the culture,or both.

[0042] In another embodiment, the present invention extends to anisolated nucleic acid molecule which encodes murine prolinedehydrogenase protein (Prodh). In particular, the present inventionextends to an isolated nucleic acid molecule comprising a DNA sequenceof SEQ ID NO:3, degenerate variants thereof, fragments thereof, oranalogs or derivatives thereof. Naturally, the present invention extendsto an isolated nucleic acid molecule which is hybridizable understandard hybridization conditions to an isolated nucleic acid moleculecomprising a DNA sequence of SEQ ID NO:3, degenerate variants thereof,fragments thereof, or analogs or derivatives thereof. As explainedabove, isolated nucleic acid molecules of the invention can bedetectably labeled. Examples of detectable labels having applicationsherein are described infra.

[0043] In addition, the present invention extends to an isolated nucleicacid molecule which encodes a Prodh protein comprising an amino acidsequence of SEQ ID NO:4, conservative variants thereof, fragmentsthereof, or analogs or derivatives thereof. A particular example of suchan isolated nucleic acid molecule comprises a DNA sequence of SEQ IDNO:3, degenerate variants thereof, fragments thereof, or analogs orderivatives thereof.

[0044] Naturally, the present invention extends to an isolated Prodhprotein comprising an amino acid sequence of SEQ ID NO:4, conservativevariants thereof, fragments thereof, or analogs or derivatives thereof.

[0045] Further, the present invention extends to an antibody having amurine proline dehydrogenase protein, a fragment thereof, a conservativevariant thereof, or an analog or derivative thereof as an immunogen. Ina particular example, the immunogen of an antibody of the invention is aprotein comprising an amino acid sequence of SEQ ID NO:4, conservativevariants thereof, fragments thereof, or analogs or derivatives thereof.Furthermore, such an antibody can be polyclonal, monoclonal or chimeric,and optionally can be detectably labeled.

[0046] The present invention further extends to cloning vectors whichcan replicate or “clone” an isolated nucleic acid molecule which encodesa murine proline dehydrogenase protein, conservative variants thereof,fragments thereof, or analogs or derivatives thereof. In one embodiment,a cloning vector of the invention comprises an origin of replication andan isolated nucleic acid molecule comprising the DNA sequence of SEQ IDNO:3, degenerate variants thereof, fragments thereof, or analogs orderivatives thereof. Another embodiment of a cloning vector of theinvention comprises an isolated nucleic acid molecule and an origin ofreplication, wherein the isolated nucleic acid molecule is hybridizableunder standard hybridization conditions to an isolated nucleic acidmolecule comprising a DNA sequence of SEQ ID NO:3, degenerate variantsthereof, fragments thereof, or analogs or derivatives thereof. Asexplained above, numerous commercially available cloning vectors whichcomprise a polylinker region have ready applications in the presentinvention. One of ordinary skill in the art can readily insert anisolated nucleic acid molecule of the invention into a commerciallyavailable cloning vector using routine recombinant DNA techniques,described infra. Particular examples of cloning vectors havingapplications herein include, but certainly are not limited to E. coli,bacteriophages, plasmids, or pUC plasmid derivatives. Furthermore,bacteriophage vectors having applications herein include lambdaderivatives, plasmids further comprise pBR322 derivatives, and pUCplasmid derivatives further comprise pGEX vectors, or pmal-c, pFLAG, toname only a few.

[0047] In addition, the present invention extends to expression vectorsfor producing a murine proline dehydrogenase protein comprising an aminoacid sequence of SEQ ID NO:4, conservative variants thereof, fragmentsthereof, or analogs or derivatives thereof. In particular, the inventionextends to an expression vector comprising an isolated nucleic acidmolecule comprising a DNA sequence of SEQ ID NO:3, degenerate variantsthereof, fragments thereof, or analogs or derivatives thereof,operatively associated with a promoter. In another embodiment, anexpression vector of the invention comprises an isolated nucleic acidmolecule operatively associated with a promoter, wherein the isolatednucleic acid molecule is hybridizable under standard hybridizableconditions to an isolated nucleic acid molecule comprising a DNAsequence of SEQ ID NO:3, degenerate variants thereof, fragments thereof,or analogs or derivatives thereof. Numerous expression vectors that arecommercially available have applications herein. Examples of readilyavailable vectors include derivatives of SV40 and known bacterialplasmids, e.g., E. coli plasmids col E1, pCR1, pBR322, pMa1-C2, pET,pGEX (Smith et al., 1988, Gene 67:31-40), pMB9 and their derivatives,plasmids such as RP4; phage DNAS, e.g., the numerous derivatives ofphage λ, e.g., NM989, and other phage DNA, e.g., M13 and filamentoussingle stranded phage DNA; yeast plasmids such as the 2μ plasmid orderivatives thereof; vectors useful in eukaryotic cells, such as vectorsuseful in insect or mammalian cells; vectors derived from combinationsof plasmids and phage DNAs, such as plasmids that have been modified toemploy phage DNA or other expression control sequences; and the like.

[0048] Insertion of an isolated nucleic acid molecule into such anexpression vector is readily within the skill of one of ordinary skillin the art using recombinant DNA techniques described herein.

[0049] Further, numerous promoters have applications herein. Examples ofsuch promoters include, but certainly are not limited to immediate earlypromoters of hCMV, early promoters of SV40, early promoters ofadenovirus, early promoters of vaccinia, early promoters of polyoma,late promoters of SV40, late promoters of adenovirus, late promoters ofvaccinia, late promoters of polyoma, the lac system the trp system, theTAC system, the TRC system, the major operator and promoter regions ofphage lambda, control regions of fd coat protein, 3-phosphoglyceratekinase promoter, acid phosphatase promoter, or promoters of yeast αmating factor, to name only a few.

[0050] The present invention further extends to unicellular hoststransformed or transfected with an expression vector of the invention.In particular, the present invention extends to a unicellular hosttransformed or transfected with an expression vector comprising anisolated nucleic acid molecule comprising a DNA sequence of SEQ ID NO:3,degenerate variants thereof, fragments thereof, or analogs orderivatives thereof, operatively associated with a promoter. In anotherembodiment, the present invention extends to a unicellular hosttransformed or transfected with an expression vector comprising anisolated nucleic acid molecule operatively associated with a promoter,wherein the isolated nucleic acid molecule is hybridizable understandard hybridization conditions to an isolated nucleic acid moleculecomprising a DNA sequence of SEQ ID NO:3, degenerate variants thereof,fragments thereof, or analogs or derivative thereof. Examples ofunicellular hosts having applications herein include but certainly arenot limited to E. coli, Pseudonomas, Bacillus, Strepomyces, yeast, CHO,R1.1, B-W, L-M, COS1, COS7, BSC1, BSC40, BMT10 or Sf9 cells.

[0051] Naturally, the present invention extends to methods of producinga murine proline dehydrogenase protein comprising an amino acid sequenceof SEQ ID NO:4, conservative variants thereof, fragments thereof, oranalogs or derivatives thereof. In one embodiment, the method comprisesthe steps of:

[0052] a) culturing a unicellular host transformed or transfected withan expression vector comprising an isolated nucleic acid moleculecomprising a DNA sequence of SEQ ID NO:3, a degenerate variant thereof,a fragment thereof, or an analog or derivative thereof, operativelyassociated with a promoter, under conditions that provide for expressionof the isolated nucleic acid molecule to produce a protein comprising anamino acid sequence of SEQ ID NO:4, conservative variant thereof,fragment thereof, or analog or derivative thereof; and

[0053] b) recovering the protein from the unicellular host, the culture,or both.

[0054] In another embodiment, the method comprises the steps of:

[0055] a) culturing a unicellular host transformed or transfected withan expression vector comprising an isolated nucleic acid moleculeoperatively associated with a promoter, wherein the isolated nucleicacid molecule is hybridizable under standard hybridization conditions toan isolated nucleic acid molecule comprising a DNA sequence of SEQ IDNO:3, degenerate variants thereof, fragments thereof, or analogs orderivatives thereof under conditions that provide for expression of theisolated nucleic acid molecule to produce a protein comprising an aminoacid sequence of SEQ ID NO:4, conservative variant thereof, fragmentthereof, or analog or derivative thereof; and

[0056] b) recovering the protein from the unicellular host, the culture,or both.

[0057] The present invention further extends to an isolated variantallele of a human proline dehydrogenase (PRODH) gene, wherein the PRODHgene comprises a DNA sequence of SEQ ID NO:1, and the variant allelecomprises a DNA sequence having at least one variation in SEQ ID NO:1,wherein the at least one variation comprises:

[0058] a G to A transition in the third position of codon 83;

[0059] a C to T transition in the first position of codon 101;

[0060] a G to A transition in the second position of codon 101;

[0061] a C to T transition in the first position of codon 247;

[0062] a C to T transition in the third position of codon 342;

[0063] a C to T transition in the third position of codon 421;

[0064] an A to G transition in the second position of codon 437;

[0065] a T to C transition in the first position of codon 497; or

[0066] a combination thereof.

[0067] Moreover, the present invention extends to an isolated nucleicacid molecule hybridizable under standard hybridization conditions to anisolated variant allele of a human PRODH gene, wherein the isolatedvariant allele comprises a DNA sequence having at least one variation inSEQ ID NO:1, and the at least one variation comprises:

[0068] a G to A transition in the third position of codon 83;

[0069] a C to T transition in the first position of codon 101;

[0070] a G to A transition in the second position of codon 101;

[0071] a C to T transition in the first position of codon 247;

[0072] a C to T transition in the third position of codon 342;

[0073] a C to T transition in the third position of codon 421;

[0074] an A to G transition in the second position of codon 437;

[0075] a T to C transition in the first position of codon 497; or

[0076] a combination thereof.

[0077] In addition, the present invention extends to a detectablylabeled isolated variant allele of a PRODH gene, wherein the PRODH genecomprises a DNA sequence of SEQ ID NO:1, and the variant allelecomprises a DNA sequence having at least one variation in SEQ ID NO:1,wherein the at least one variation comprises:

[0078] a G to A transition in the third position of codon 83;

[0079] a C to T transition in the first position of codon 101;

[0080] a G to A transition in the second position of codon 101;

[0081] a C to T transition in the first position of codon 247;

[0082] a C to T transition in the third position of codon 342;

[0083] a C to T transition in the third position of codon 421;

[0084] an A to G transition in the second position of codon 437;

[0085] a T to C transition in the first position of codon 497; or

[0086] a combination thereof.

[0087] Numerous detectable labels have applications in the presentinvention. For example the detectable label can be a radioactiveelement, such as the isotopes ³H, ¹⁴C, ³²P, ³⁵S, ³⁶Cl, ⁵¹Cr, ⁵⁷Co, ⁵⁸Co,⁵⁹Fe, ⁹⁰Y, ¹²⁵I, ¹³¹I, and ¹⁸⁶Re, to name only a few. Chemicals whichfluoresce, or enzymes such as alkaline phosphatase or horseradishperoxidase, can also be used as detectable labels.

[0088] Moreover, the present invention extends to a detectably labeledisolated nucleic acid molecule hybridizable under standard hybridizationconditions to an isolated variant allele of a PRODH gene, wherein thePRODH gene comprises a DNA sequence of SEQ ID NO:1, and the variantallele comprises a DNA sequence having at least one variation in SEQ IDNO:1, wherein the at least one variation comprises:

[0089] a G to A transition in the third position of codon 83;

[0090] a C to T transition in the first position of codon 101;

[0091] a G to A transition in the second position of codon 101;

[0092] a C to T transition in the first position of codon 247;

[0093] a C to T transition in the third position of codon 342;

[0094] a C to T transition in the third position of codon 421;

[0095] an A to G transition in the second position of codon 437;

[0096] a T to C transition in the first position of codon 497; or

[0097] a combination thereof.

[0098] Detectable labels set forth throughout the specification haveapplications in such an isolated nucleic acid molecule.

[0099] In addition, the present invention extends to an isolated variantallele of a PRODH gene which encodes a variant human proline PRODHcomprising at least one variation in the amino acid sequence of PRODH,wherein PRODH comprises an amino acid sequence of SEQ ID NO:2, and isencoded by a human proline dehydrogenase gene comprising a DNA sequenceof SEQ ID NO:1. A variant PRODH protein of the present inventioncomprises an amino acid sequence having at least one variation in SEQ IDNO:2, wherein the at least one variation comprises:

[0100] Arg101Trp;

[0101] Arg101Glu;

[0102] Glu437Arg; or

[0103] a combination thereof.

[0104] In particular, a variant allele of a human PRODH gene comprisinga DNA sequence comprising a C to T transition in the first position ofcodon 101 of SEQ ID NO:1, encodes a variant human PRODH proteincomprising an amino acid sequence comprising an Arg101Trp variation inSEQ ID NO:2. Furthermore, a variant allele of a human PRODH genecomprising a DNA sequence having a G to A transition in the secondposition of codon 101 of SEQ ID NO:1 encodes a variant human PRODHprotein comprising an amino acid sequence having an Arg101Glu variationin SEQ ID NO:2. Hence naturally, a variant allele of a human PRODH genecomprising a DNA sequence having an A to G transition in the secondposition of codon 437 of SEQ ID NO:1 encodes a variant human PRODHprotein comprising an amino acid sequence having a Glu437Arg variationin SEQ ID NO:2. As explained above, the present invention also extendsto a variant allele of a human PRODH gene comprising a combination ofvariations set forth herein, which encode a variant human PRODH proteincomprising an amino acid sequence having a combination of amino acidresidue variations in SEQ ID NO:2 as described above.

[0105] Naturally, the present invention extends to an isolated varianthuman PRODH protein comprising an amino acid sequence having at leastone variation in SEQ ID NO:2, wherein the at least one variationcomprises:

[0106] Arg101Trp;

[0107] Arg101Glu;

[0108] Glu437Arg; or

[0109] a combination thereof.

[0110] Furthermore, the present invention extends to an antibody havinga variant proline dehydrogenase protein of the present invention as animmunogen. Such an antibody can be a polyclonal antibody, a monoclonalantibody, or a chimeric antibody. Moreover, an antibody of the presentinvention can be detectably labeled. Examples of detectable labels whichhave applications in this embodiment comprises a radioactive element, achemical which fluoresces, or an enzyme, to name only a few.

[0111] In addition, the present invention extends to cloning vectorsthat can be used to clone copies of a variant allele of a PRODH gene ofthe present invention. An example of such a cloning vector comprises anorigin of replication and an isolated variant allele of a human PRODHgene, wherein the PRODH gene comprises a DNA sequence of SEQ ID NO:1,and an isolated variant allele of the PRODH gene comprises a DNAsequence having at least one variation in SEQ ID NO:1 wherein the atleast one variation comprises:

[0112] a G to A transition in the third position of codon 83;

[0113] a C to T transition in the first position of codon 101;

[0114] a G to A transition in the second position of codon 101;

[0115] a C to T transition in the first position of codon 247;

[0116] a C to T transition in the third position of codon 342;

[0117] a C to T transition in the third position of codon 421;

[0118] an A to G transition in the second position of codon 437;

[0119] a T to C transition in the first position of codon 497; or

[0120] a combination thereof.

[0121] Moreover, the present invention extends to a cloning vectorcomprising an origin of replication and an isolated nucleic acidmolecule hybridizable under standard hybridization conditions to anisolated variant allele of a PRODH gene, wherein the PRODH genecomprises a DNA sequence of SEQ ID NO:1, and the isolated variant alleleof the PRODH gene comprises a DNA sequence having at least one variationin SEQ ID NO:1 wherein the at least one variation comprises:

[0122] a G to A transition in the third position of codon 83;

[0123] a C to T transition in the first position of codon 101;

[0124] a G to A transition in the second position of codon 101;

[0125] a C to T transition in the first position of codon 247;

[0126] a C to T transition in the third position of codon 342;

[0127] a C to T transition in the third position of codon 421;

[0128] an A to G transition in the second position of codon 437;

[0129] a T to C transition in the first position of codon 497; or

[0130] a combination thereof.

[0131] Numerous cloning vectors have applications in the presentinvention and are readily available to a skilled artisan. Furthermore,it is well within the knowledge of one of ordinary skill in the art toinsert an isolated PRODH variant allele of the present invention into acommercially available cloning vector using recombinant DNA techniquesdescribed infra. Examples of a cloning vector having applications in thepresent invention include E. coli, bacteriophages, such as lambdaderivatives, plasmids, such as pBR322 derivatives, and pUC plasmidderivatives, such as pGEX vectors, or pmal-c, pFLAG, to name only a few.

[0132] The present invention further extends to a unicellular hosttransformed or transfected with a cloning vector which comprises anisolated variant allele of the human PRODH gene as described above.Examples of hosts which are readily available to the skilled artisan andcan be transformed or transfected with a cloning vector of the presentinvention include, but are not limited to E. coli, Pseudonomas,Bacillus, Strepomyces, yeast, CHO, R1.1, B-W, L-M, COS1, COS7, BSC1,BSC40, BMT10 or Sf9 cells.

[0133] The present invention further extends to a method of cloning orproducing “copies” of an isolated variant allele of a human PRODH geneof the invention, which comprises inserting a cloning vector into aunicellular host, and then inducing the host to self replicate. Duringthe self replication of the host, the origin of replication of thecloning vector causes the replication of the cloning vector. After theunicellular host has self replicated numerous times, the cloning vectorscan be isolated from the cloned host, and the isolated nucleic acidmolecule can be isolated via restriction digestion from the cloningvectors.

[0134] Naturally, the present invention extends to expression vectorscomprising an isolated variant allele of a PRODH gene operativelyassociated with a promoter, wherein the PRODH gene comprises a DNAsequence of SEQ ID NO:1, and an isolated variant allele of the presentinvention comprises a DNA sequence having at least one variation in SEQID NO:1 wherein the at least one variation comprises:

[0135] a G to A transition in the third position of codon 83;

[0136] a C to T transition in the first position of codon 101;

[0137] a G to A transition in the second position of codon 101;

[0138] a C to T transition in the first position of codon 247;

[0139] a C to T transition in the third position of codon 342;

[0140] a C to T transition in the third position of codon 421;

[0141] an A to G transition in the second position of codon 437;

[0142] a T to C transition in the first position of codon 497; or

[0143] a combination thereof.

[0144] Furthermore, the present invention extends to an expressionvector comprising an isolated nucleic acid molecule operativelyassociated with a promoter, wherein the isolated nucleic acid moleculeis hybridizable under standard hybridization conditions to an isolatedvariant allele of a PRODH gene, wherein the PRODH gene comprises a DNAsequence of SEQ ID NO:1, and an isolated variant allele of the presentinvention comprises a DNA sequence having at least one variation in SEQID NO:1 wherein the at least one variation comprises:

[0145] a G to A transition in the third position of codon 83;

[0146] a C to T transition in the first position of codon 101;

[0147] a G to A transition in the second position of codon 101;

[0148] a C to T transition in the first position of codon 247;

[0149] a C to T transition in the third position of codon 342;

[0150] a C to T transition in the third position of codon 421;

[0151] an A to G transition in the second position of codon 437;

[0152] a T to C transition in the first position of codon 497; or

[0153] a combination thereof.

[0154] Numerous promoters which are readily available to a skilledartisan, have applications in any expression vector of the invention.For example, immediate early promoters of hCMV, early promoters of SV40,early promoters of adenovirus, early promoters of vaccinia, earlypromoters of polyoma, late promoters of SV40, late promoters ofadenovirus, late promoters of vaccinia, late promoters of polyoma, thelac system, the trp system, the TAC system, the TRC system, the majoroperator and promoter regions of phage lambda, control regions of fdcoat protein, 3-phosphoglycerate kinase promoter, acid phosphatasepromoter, or promoters of yeast α mating factor, to name only a few,have applications herein. Furthermore, it is well within the knowledgeof one of ordinary skill in the art to insert an isolated variant alleleof the invention, or an isolated nucleic acid molecule hybridizableunder standard hybridization conditions to an isolated variant allele ofthe invention into a commercially available expression vector, usingrecombinant DNA techniques described infra.

[0155] In addition, the present invention extends to a unicellular hosttransformed or transfected with an expression vector of the presentinvention. Examples of hosts which can be transformed or transfectedwith an expression vector of the present invention, and haveapplications in the present invention, include, but are not limited to,E. coli, Pseudonomas, Bacillus, Strepomyces, yeast, CHO, R1.1, B-W, L-M,COS1, COS7, BSC1, BSC40, BMT10or Sf9 cells.

[0156] Naturally, the present invention extends to a method forproducing a variant human PRODH protein comprising an amino acid havingat least one variation in SEQ ID NO:2, wherein the at least onevariation comprises

[0157] Arg101Trp;

[0158] Arg101Glu;

[0159] Glu437Arg; or

[0160] a combination thereof.

[0161] An example of such a method comprises the steps of culturing aunicellular host transformed or transfected with an expression vectorcomprising an isolated variant allele of a PRODH gene operativelyassociated with a promoter, wherein the isolated variant allele of thePRODH gene comprises a DNA sequence having at least one variation in SEQID NO:1, and the at least one variation comprises:

[0162] a G to A transition in the third position of codon 83;

[0163] a C to T transition in the first position of codon 101;

[0164] a G to A transition in the second position of codon 101;

[0165] a C to T transition in the first position of codon 247;

[0166] a C to T transition in the third position of codon 342;

[0167] a C to T transition in the third position of codon 421;

[0168] an A to G transition in the second position of codon 437;

[0169] a T to C transition in the first position of codon 497; or

[0170] a combination thereof,

[0171] under conditions that provide for expression of the variantallele. The variant PRODH protein produced from such expression is thenrecovered from the unicellular host, the culture, or both.

[0172] Yet another method of the present invention for producing avariant PRODH protein involves culturing a unicellular host transformedor transfected with an expression vector comprising an isolated nucleicacid molecule operatively associated with a promoter, wherein theisolated nucleic acid molecule is hybridizable under standardhybridization conditions to an isolated variant allele of a PRODH genecomprising a DNA sequence having at least one variation in SEQ ID NO:1,wherein the at least one variation comprises:

[0173] a G to A transition in the third position of codon 83;

[0174] a C to T transition in the first position of codon 101;

[0175] a G to A transition in the second position of codon 101;

[0176] a C to T transition in the first position of codon 247;

[0177] a C to T transition in the third position of codon 342;

[0178] a C to T transition in the third position of codon 421;

[0179] an A to G transition in the second position of codon 437;

[0180] a T to C transition in the first position of codon 497; or

[0181] a combination thereof,

[0182] under conditions that provide for expression of the isolatednucleic acid molecule. The variant human PRODH protein produced fromsuch induced expression is then recovered from the unicellular host, theculture, or both.

[0183] Furthermore, the present invention extends to a method fordetecting a susceptibility to, or the presence of schizophrenia or adisease or disorder related thereto, such as obsessive compulsivedisorder (OCD), bipolar disorder (BP) or major depressive disorder in asubject, wherein the method comprises measurement of the levels ofactivity of an enzyme in a bodily sample which is involved in prolinecatabolism. A comparison of the measurement of the levels of activity ofthe enzyme in the bodily sample is then made with the levels of activityof the enzyme in a standard. A modulated level of enzyme activity in thesample relative to the level of activity in the standard is indicativeof a susceptibility to, or the presence of, schizophrenia or a diseaseor disorder related thereto, such as obsessive compulsive disorder(OCD), bipolar disorder (BP) or major depressive disorder, in thesubject. In a particular embodiment, the enzyme involved in prolinecatabolism is proline dehydrogenase (PRODH), and a reduced level ofactivity of PRODH in a bodily sample from the subject compared to thelevel of PRODH activity in the standard is indicative of increasedsusceptibility to, or the presence of schizophrenia or a disease ordisorder related thereto in the subject relative to the susceptibilityof the standard. Methods of assaying activity of proline dehydrogenasein a bodily sample are readily available to the skilled artisan.

[0184] The present invention further extends to a method for determininga susceptibility to, or the presence of schizophrenia or a disease ordisorder related thereto in a subject, such as obsessive compulsivedisorder (OCD), bipolar disorder (BP) or major depressive disorder(MDD), wherein the method comprises the steps of:

[0185] a) removing a bodily sample from the subject, wherein the samplecomprises a PRODH gene; and

[0186] b) determining whether the PRODH gene of the bodily samplecomprises a DNA sequence having a variation in SEQ ID NO:1 comprising aT to C transition in the first position of codon 497. The presence ofthe variant allele the PRODH gene in a bodily sample of the subjectindicates the subject has an increased susceptibility to schizophreniaor a disease or disorder related thereto, such as obsessive compulsivedisorder (OCD), bipolar disorder (BP) or major depressive disorder (MDD)relative to the susceptibility of a standard, wherein the bodily sampleof the standard comprises a PRODH gene comprising a DNA sequence of SEQID NO:1.

[0187] Furthermore, the present invention extends to an assay forscreening drugs and other agents for ability to treat schizophrenia or adisease or disorder related thereto. Such an assay of the presentinvention comprises the steps of culturing an observable cellular testcolony which produces PRODH and which has been inoculated with the drugor agent to be assayed, harvesting a cellular extract from the cellulartest colony, and determining the level of activity of PRODH in the testcolony. An increase or decrease in the level of activity of PRODH inthis test colony compared to a control test colony not inoculated withthe drug, or compared to the level of activity of PRODH in the cellulartest colony prior to inoculation with the drug or agent, is indicativeof the ability of the drug or agent to modulate the production,stability, degradation or activity of PRODH, which in turn is indicativeof the drug or agent's ability to treat schizophrenia or a disease ordisorder related thereto, such as obsessive compulsive disorder (OCD),bipolar disorder (BP) or major depressive disorder. An increase in thelevel of activity of PRODH in the test colony after inoculation with thedrug or agent compared to the level of activity in the control colony,or in the cellular test colony prior to inoculation with the drug oragent, indicates the drug has the ability to be used to treatschizophrenia or a disorder related thereto.

[0188] In another embodiment, the present invention extends to an assaysystem which may be prepared in the form of a test kit for thequantitative analysis of the extent of the presence and/or activity ofPRODH, or to identify drugs or other agents that may potentiate orincrease such activity. Broadly, a system or test kit of the presentinvention may comprise a labeled component prepared by one of theradioactive and/or enzymatic techniques discussed herein, coupling thelabel to PRODH, its agonists and/or antagonists, and one or moreadditional immunochemical reagents, at least one of which is a free orimmobilized ligand, capable either of binding with the labeledcomponent, its binding partner, one of the components to be determined,or their binding partner(s). The system or test kit may also comprise apolymerase chain reaction based (PCR) assay which can be used toquantify the PRODH levels of a sample.

[0189] Hence, the present invention extends to a test kit to facilitatediagnosis and treatment of schizophrenia or a disease or disorderrelated thereto, such as obsessive compulsive disorder (OCD), bipolardisorder (BP) or major depressive disorder, comprising:

[0190] (a) a predetermined amount of a detectably labeled specificbinding partner of a PRODH protein;

[0191] (b) other reagents; and

[0192] (c) directions for use of said kit.

[0193] Examples of the labeled immunochemically reactive component ofsuch a test kit can be selected from the group consisting of polyclonalantibodies to PRODH, monoclonal antibodies to PRODH, chimeric antibodiesto PRODH, fragments of such antibodies, and mixtures of such antibodies.

[0194] Furthermore, the present invention extends to a test kit tofacilitate diagnosis and treatment of schizophrenia or a disease ordisorder related thereto in a subject, wherein the test kit comprises:

[0195] (a) PCR oligonucleotide primers suitable to detecting a variantallele of the PRODH gene in a sample;

[0196] (b) other reagents; and

[0197] (c) directions for use of the kit.

[0198] The present invention further extends to a test kit to facilitatediagnosis and treatment of schizophrenia or a disease or disorderrelated thereto in a eukaryotic cellular sample, wherein the test kitcomprises:

[0199] (a) PCR oligonucleotide primers suitable for detection of anisolated variant allele of a PRODH gene, wherein the PRODH genecomprises a DNA sequence of SEQ ID NO:1, and the isolated variantcomprises a DNA sequence comprising a T to C transition in the firstposition of codon 497 of SEQ ID NO:1;

[0200] (b) other reagents; and

[0201] (c) directions for use of the kit.

[0202] In another embodiment, the present invention extends to treatingschizophrenia or a disease or disease or disorder related thereto in asubject, such as obsessive compulsive disorder (OCD), bipolar disorder(BP) or major depressive disorder. An example of such a method comprisesadministering to the subject a therapeutically effective amount of acomposition comprising PRODH, wherein PRODH comprises an amino acidsequence of SEQ ID NO:2, conservative variants thereof, fragmentsthereof, or analogs or derivatives thereof. Optionally, the compositionof the invention can be administered alone or in combination withadditional therapeutic agents to treat the subject.

[0203] In addition, the present invention extends to a method fordetermining the schizophrenic-related pharmacological activity of anagent, wherein the method comprises the steps of:

[0204] administering the agent to a mammal;

[0205] determining the level of activity of PRODH in the mammal; and

[0206] comparing the level of activity of PRODH in the mammal to thelevel of activity of PRODH in a control mammal to which the agent wasnot administered. An increase in the level of activity of PRODH in themammal relative to activity of PRODH in the control mammal indicates theagent has a schizophrenic-related pharmacological activity, andpotential as a therapeutic agent for treating schizophrenia or a diseaseor disorder related thereto, such as obsessive compulsive disorder(OCD), bipolar disorder (BP) or major depressive disorder.

[0207] In yet another aspect, the present invention extends to a methodfor determining the schizophrenic-related pharmacological activity of anagent, wherein the method comprises the steps of:

[0208] determining a basal level of activity of PRODH in the mammal;

[0209] administering the agent to the mammal;

[0210] determining the level of activity of PRODH in the mammal afteradministration of the agent; and

[0211] comparing the level of activity of PRODH after administration ofthe agent to the basal level of activity.

[0212] An increase in the level of activity of PRODH in the mammalrelative to the basal level in the mammal indicates the compound has aschizophrenic-related pharmacological activity, and may have potentialas a therapeutic agent for treating schizophrenia or a disease ordisorder related thereto, such as obsessive compulsive disorder (OCD),bipolar disorder (BP) or major depressive disorder.

[0213] What's more, the present invention extends to an isolated variantallele of the Prodh gene which encodes a mutated murine Prodh protein.In particular, the present invention extends to an isolated variantallele of the Prodh gene, wherein the isolated variant allele comprisesa DNA sequence of FIG. 10 (SEQ ID NO:7), degenerate variants thereof,fragments thereof, or analogs or derivatives thereof.

[0214] The present invention also extends to an isolated nucleic acidmolecule hybridzable under standard hybridization conditions to theisolated variant allele of the murine Prodh gene comprising a DNAsequence of SEQ ID NO:7, degenerate variants thereof, fragments thereof,or analogs or derivatives thereof.

[0215] In addition, the present invention extends to an isolated variantallele of the Prodh gene which comprises a DNA sequence of SEQ ID NO:7,degenerate variants thereof, fragments thereof, or analogs orderivatives thereof, detectably labeled. Naturally, the presentinvention extends to an isolated nucleic acid molecule detectablylabeled, wherein the isolated nucleic acid molecule is hybridizableunder standard hybridization conditions to an isolated variant allele ofthe Prodh gene, wherein the isolated variant allele comprises a DNAsequence of SEQ ID NO:7, degenerate variants thereof, fragments thereof,or analogs or derivatives thereof.

[0216] Moreover, the present invention extends to an isolated nucleicacid molecule encoding an isolated mutant murine Prodh proteincomprising an amino acid sequence of FIG. 11 (SEQ ID NO:8), conservativevariants thereof, fragments thereof, or analogs or derivatives thereof.

[0217] Naturally, the present invention extends to an isolated Prodhcomprising an amino acid sequence of SEQ ID NO:8, conservative variantsthereof, fragments thereof, or analogs or derivatives thereof.

[0218] Also, the present invention extends to an antibody having anisolated mutant Prodh of the invention, conservative variants thereof,fragments thereof, or analogs or derivatives thereof as an immunogen.Such an antibody can be polyclonal, monoclonal, or chimeric. Further,such an antibody can be detectably labeled. As explained above, numerousexamples of detectable labels having applications in an antibody of theinvention are described infra.

[0219] In addition, the present invention extends to cloning vectors forcreating copies or “cloning” an isolated variant allele of a Prodh geneof the invention, degenerate variants thereof, fragments thereof, oranalogs or derivatives thereof. In particular, the present inventionextends to a cloning vector comprising an isolated variant allele of aProdh gene, wherein the isolated variant allele comprises a DNA sequenceof SEQ ID NO:7, degenerate variants thereof, fragments thereof, oranalogs or derivatives thereof, and an origin of replication. In anotherembodiment, the invention extends to a cloning vector comprising anorigin of replication and an isolated nucleic acid molecule hybridizableunder standard hybridization conditions to an isolated variant allele ofa Prodh gene, wherein the isolated variant allele comprises a DNAsequence of SEQ ID NO:7, degenerate variants thereof, fragments thereof,or analogs or derivatives thereof.

[0220] Numerous cloning vectors which are commercially available to theskilled artisan, and can be used as a cloning vector for an isolatedvariant allele of a Prodh gene. Examples of such cloning vectors, androutine recombinant DNA techniques to produce such a vector aredescribed infra.

[0221] Naturally, the present invention extends to an expression vectorfor expressing an isolated variant allele of a murine Prodh gene,degenerate variants thereof, fragments thereof, or analogs orderivatives thereof, along with an isolated nucleic acid moleculehybridizable thereto under standard hybridization conditions, to producea mutated murine Prodh protein, conservative variants thereof, fragmentsthereof, or analogs or derivatives thereof. In particular, an expressionvector of the invention comprises an isolated variant allele of themurine Prodh gene, wherein the isolated variant allele comprises a DNAsequence of SEQ ID NO:7, degenerate variants thereof, fragments thereof,or analogs or derivatives thereof, operatively associated with apromoter. In another embodiment, an expression vector of the inventioncomprises an isolated nucleic acid molecule operatively associated witha promoter, wherein the isolated nucleic acid molecule is hybridzableunder standard hybridization conditions to an isolated variant allele ofthe Prodh gene, wherein the isolated variant allele comprises a DNAsequence of SEQ ID NO:7, degenerate variants thereof, fragments thereof,or analogs or derivatives thereof.

[0222] Numerous expression vectors can be used to express the isolatedvariant allele of the Prodh gene, degenerate variants thereof, fragmentsthereof, or analogs or derivatives thereof, or an isolated nucleic acidmolecule hybridizable to the isolated variant allele under standardhybridization conditions. In particular, such expression vectors aregenerally commercially available to the skilled artisan, and likecloning vectors, comprise polylinker sites. As a result, commerciallyavailable expression vectors can be manipulated in a similar fashion inwhich cloning vectors of the invention are manipulated. Hence a skilledartisan can readily insert the isolated variant allele of the Prodhgene, degenerate variants thereof, fragments thereof, or analogs orderivatives thereof, or an isolated nucleic acid molecule hybridizablethereto under standard hybridization conditions into an expressionvector such that the isolated variant allele or an isolated nucleic acidmolecule hybridizable thereto under standard hybridization conditions isoperatively associated with a promoter. Examples of expression vectorshaving applications herein are described infra.

[0223] Moreover, the present invention extends to a unicellular hosttransformed or transfected with an expression vector comprising anisolated variant allele of the Prodh gene, operatively associated with apromoter, wherein the isolated variant allele encodes a mutant murineproline dehydrogenase protein comprising an amino acid sequence of SEQID NO:8, conservative variants thereof, fragments thereof, or analogs orderivatives thereof. In one embodiment, the present invention extends toa unicellular host transformed or transfected with an expression vectorcomprising an isolated variant allele of the Prodh gene, operativelyassociated with a promoter, wherein the isolated variant allelecomprises a DNA sequence of SEQ ID NO:7, degenerate variants thereof,fragments thereof, or analogs or derivatives thereof. In anotherembodiment, the invention extends to a unicellular host transformed ortransfected with an expression vector comprising an isolated nucleicacid molecule operatively associated with a promoter, wherein theisolated nucleic acid molecule is hybridizable under standardhybridization conditions to an isolated variant allele of the Prodhgene, wherein the isolated variant allele comprises a DNA sequence ofSEQ ID NO:7, a degenerate variant thereof, a fragment thereof, or ananalog or derivative thereof. Numerous unicellular hosts which arereadily available to the skilled artisan have applications in thepresent invention.

[0224] Naturally, the present invention extends to method for producinga mutant murine proline dehydrogenase protein comprising an amino acidsequence of SEQ ID NO:8, conservative variants thereof, fragmentsthereof, or analogs or derivatives thereof. In one embodiment, a methodfor producing a mutant murine proline dehydrogenase comprises the stepsof:

[0225] a) culturing a unicellular host of transformed or transfectedwith an expression vector comprising an isolated variant allele of theprodh gene, wherein the isolated variant allele comprises a DNA sequenceof SEQ ID NO:7, a degenerate variant thereof, a fragment thereof, or ananalog or derivative thereof, operatively associated with a promoter,under conditions that provide for expression of the isolated variantallele to produce a mutant murine proline dehydrogenase proteincomprising an amino acid sequence of SEQ ID NO:8, a conservative variantthereof, a fragment thereof, or analog or derivative thereof; and

[0226] b) recovering the protein from the unicellular host, the culture,or both.

[0227] In another embodiment, the method comprises the steps of:

[0228] a) culturing a unicellular host of transformed or transfectedwith an expression vector comprising an isolated nucleic acid moleculehybridizable under standard hybridization conditions to an isolatedvariant allele of the Prodh gene, wherein the isolated variant allelecomprises a DNA sequence of SEQ ID NO:7, a degenerate variant thereof, afragment thereof, or an analog or derivative thereof, operativelyassociated with a promoter, under conditions that provide for expressionof the isolated nucleic acid molecule, to produce a protein comprisingan amino acid sequence of SEQ ID NO:8, a conservative variant thereof, afragment thereof, or an analog or derivative thereof, and

[0229] b) recovering the protein from the unicellular host, the culture,or both.

[0230] Furthermore, the present invention extends to a method foridentifying a drug or agent for treating schizophrenia or a disease ordisorder related thereto. An example of such a method comprises thesteps of:

[0231] performing an first pre-pulse inhibition test (PPI) test on amouse having within its genome two copies of an isolated variant alleleof a Prodh gene comprising a DNA sequence of SEQ ID NO:7, wherein bothcopies are capable of expressing a mutant Prodh comprising an amino acidsequence of SEQ ID NO:8, to obtain a first percentage of inhibition ofstartle response;

[0232] administering the potential drug or agent to the mouse;

[0233] performing a second PPI test on the mouse to obtain a secondpercentage of inhibition of startle response; and

[0234] comparing the first percentage to the inhibition of startleresponse with the second percentage of startle response,

[0235] wherein an increase in percentage of inhibition in the secondpercentage of inhibition relative to the first percentage of inhibitionis indicative of the ability of the drug or agent to treat schizophreniaor a disease or disorder related thereto. Thus, if the percentage ofinhibition of startle response in the mouse having within its two activecopies of an isolated variant allele of a Prodh gene comp mouse afteradministration of the drug or agent is greater than the percentage ofinhibition of startle response in the Pro/Re mouse prior to inhibition,then the drug or agent has the ability to treat schizophrenia or adisease or disorder related thereto, such as obsessive compulsivedisorder (OCD), bipolar disorder (BP) or major depressive disorder.

[0236] Also, the present invention extends to a method for identifying adrug or agent for treating schizophrenia or a disease or disorderrelated thereto. An example of such a method comprises the steps of:

[0237] performing an first pre-pulse inhibition test (PPI) test on an F3generation mouse from a cross Pro/Re X C57B1/6J wild-type, wherein theF3 generation mouse has two copies within its genome of an isolatedvariant allele of a Prodh gene comprising a DNA sequence of SEQ ID NO:7which are capable of expressing a mutant Prodh comprising an amino acidsequence of SEQ ID NO:8, to obtain a first percentage of inhibition ofstartle response;

[0238] administering the potential drug or agent to the F3 generationmouse from a cross of Pro/Re X C57B1/6J wild-type;

[0239] performing a second PPI test on the F3 generation mouse from across of Pro/Re X C57B1/6J wild-type to obtain a second percentage ofinhibition of startle response; and

[0240] comparing the first percentage to the inhibition of startleresponse with the second percentage of startle response,

[0241] wherein an increase in percentage of inhibition in the secondpercentage of inhibition relative to the first percentage of inhibitionis indicative of the ability of the drug or agent to treat schizophreniaor a disease or disorder related thereto.

[0242] What's more, the present invention extends to a method foridentifying a drug or agent for use in treating schizophrenia or adisease or disorder related thereto, comprising the steps of:

[0243] a) administering the drug or agent to an F3 generation mouse froma cross of Pro/Re X C57B1/6J wild-type, wherein the F3 generation mousehas two copies within its genome of an isolated variant allele of aProdh gene comprising a DNA sequence of SEQ ID NO:7 which are capable ofexpressing a mutant Prodh comprising an amino acid sequence of SEQ IDNO:8;

[0244] b) performing a PPI test on the F3 generation mouse from a crossof Pro/Re X C57B1/6J wild-type to obtain a percentage of inhibition ofthe startle response in the F3 generation mouse from a cross of Pro/Re XC57B1/6J wild-type which was administered the drug or agent; and

[0245] c) comparing the percentage of inhibition of the startle responsein the F3 generation mouse from a cross of Pro/Re X C57B1/6J wild-typewith the percentage of inhibition of the startle response in anunmedicated F3 generation mouse from a cross of Pro/Re X C57B1/6Jwild-type, wherein the F3 generation mouse has two copies within itsgenome of an isolated variant allele of a Prodh gene comprising a DNAsequence of SEQ ID NO:7 which are capable of expressing a mutant Prodhcomprising an amino acid sequence of SEQ ID NO:8.

[0246] An increase in percentage of inhibition in the percentage ofinhibition in the medicated mouse relative to the percentage ofinhibition in the unmedicated mouse is indicative of the ability of thedrug or agent to treat schizophrenia or a disease or disorder relatedthereto.

[0247] Furthermore, the present invention extends to a method foridentifying a drug or agent for use in treating schizophrenia or adisease or disorder related thereto, comprising the steps of:

[0248] a) administering the drug or agent to a mouse having within itsgenome two copies of an isolated variant allele of a Prodh genecomprising a DNA sequence of SEQ ID NO:7, wherein both copies arecapable of expressing a mutant Prodh comprising an amino acid sequenceof SEQ ID NO:8;

[0249] b) performing a PPI test on the mouse to obtain a percentage ofinhibition of the startle response in the mouse; and

[0250] c) comparing the percentage of inhibition of the startle responsein the mouse with the percentage of inhibition of the startle responsein an unmedicated mouse having within its genome two copies of anisolated variant allele of a Prodh gene comprising a DNA sequence of SEQID NO:7, wherein both copies are capable of expressing a mutant Prodhcomprising an amino acid sequence of SEQ ID NO:8.

[0251] An increase in percentage of inhibition in the percentage ofinhibition in the medicated mouse relative to the percentage ofinhibition in the unmedicated mouse is indicative of the ability of thedrug or agent to treat schizophrenia or a disease or disorder relatedthereto.

[0252] In addition, the present invention extends to a method foridentifying a drug or agent for use in treating schizophrenia or adisease or disorder related thereto, comprising the steps of:

[0253] a) administering the drug or agent to a mouse having within itsgenome two copies of an isolated variant allele of a Prodh genecomprising a DNA sequence of SEQ ID NO:7, wherein both copies arecapable of expressing a mutant Prodh comprising an amino acid sequenceof SEQ ID NO:8;

[0254] b) performing a PPI test on the mouse to obtain a percentage ofinhibition of the startle response in the mouse; and

[0255] c) comparing the percentage of inhibition of the startle responsein the mouse with the percentage of inhibition of the startle responsein an unmedicated mouse having within its genome two copies of anisolated Prodh gene comprising a DNA sequence of SEQ ID NO:3, whereinboth copies are capable of expressing a Prodh comprising an amino acidsequence of SEQ ID NO:4.

[0256] If the percentage of inhibition of the startle response in themedicated mouse is statistically equivalent to the percentage ofinhibition in the mouse capable of expressing Prodh comprising a DNAsequence of SEQ ID NO:4, then the drug or agent has the ability to treatschizophrenia or a disease or disorder related thereto.

[0257] In another embodiment, the present invention extends to an amethod for identifying a drug or agent for use in treating schizophreniaor a disease or disorder related thereto, comprising the steps of:

[0258] a) administering the drug or agent to an F3 generation mouse froma cross of Pro/Re X C57B1/6J wild-type, wherein the F3 generation mousehas two copies within its genome of an isolated variant allele of aProdh gene comprising a DNA sequence of SEQ ID NO:7 which are capable ofexpressing a mutant Prodh comprising an amino acid sequence of SEQ IDNO:8;

[0259] b) performing a PPI test on the F3 generation mouse from a crossof Pro/Re X C57B1/6J wild-type administered the drug or agent to obtaina percentage of inhibition of the startle response in the mouse; and

[0260] c) comparing the percentage of inhibition of the startle responsein F3 generation mouse from a cross of Pro/Re X C57B1/6J wild-typeadministered the drug with the percentage of inhibition of the startleresponse in an F3 generation mouse from a cross of Pro/Re X C57B1/6Jwild-type, wherein the F3 generation mouse has two copies within itsgenome of an isolated Prodh gene comprising a DNA sequence of SEQ IDNO:3 which are capable of expressing a Prodh comprising an amino acidsequence of SEQ ID NO:4.

[0261] If the percentage of inhibition of the startle response in themedicated mouse is statistically equivalent to the percentage ofinhibition in the mouse capable of expressing Prodh comprising a DNAsequence of SEQ ID NO:4, then the drug or agent has the ability to treatschizophrenia or a disease or disorder related thereto.

[0262] The PPI Test is Described infra.

[0263] Accordingly it is an object of the invention to provide the DNAsequences of murine and human proline dehydrogenase genes, and the aminoacid sequences of murine and human proline dehydrogenase.

[0264] It is another object of the present invention to provideheretofore unknown variant alleles of the human PRODH gene, which can beused to map the locus of the human PRODH gene.

[0265] It is another object of the invention to provide a heretoforeunknown variant allele of the human PODH gene which is a marker for asusceptibility to, or the presence of schizophrenia or a disease ordisorder related thereto in a subject, such as obsessive compulsivedisorder (OCD), bipolar disorder (BP) or major depressive disorder in asubject.

[0266] It is yet another object of the present invention to provideisolated nucleic acid molecules, optionally detectably labeled, whichare hybridizable under standard hybridization conditions to variantalleles of the PRODH gene disclosed herein.

[0267] It is yet another object of the present invention to providevariant PRODH proteins, produced from the expression of a variantalleles of the human PRODH gene, or isolated nucleic acid moleculeshybridizable to such variant alleles under standard hybridizationconditions.

[0268] It is yet another object of the present invention to provideantibodies, optionally detectably labeled, having a variant PRODHprotein of the present invention as an immunogen, wherein suchantibodies may be polyclonal, monoclonal or chimeric.

[0269] It is yet another object of the present invention to providecommercial test kits for attending medical professionals to determinethe presence of a variant allele of the PRODH gene in a bodily sampletaken from a subject. The results of such testing can then be used todetermine the subject's susceptibility to suffer from schizophrenia or adisease or disorder related thereto, such as obsessive compulsivedisorder (OCD), bipolar disorder (BP) or major depressive disorder, orto diagnose such a disease or disorder.

[0270] It is yet another object of the present invention to provide amethod and associated assay system for screening subjects in order todetermine their susceptibility to schizophrenia or a disease or disorderrelated thereto, and to likewise select an appropriate course of therapytherefor.

[0271] It is yet another object of the present invention to providecompositions such as drugs, agents and the like, potentially effectivein either potentiating the effects of PRODH, or increasing levels ofPRODH in mammalian, especially human patients.

[0272] It is still yet another object of the present invention toprovide a method for the treatment of mammals to modulate the amount oractivity of PRODH or subunits thereof in the mammal, so as to alter theadverse consequences of diminished levels of PRODH, which can result inschizophrenia or a disease or disorder related thereto, such asobsessive compulsive disorder (OCD), bipolar disorder (BP) or majordepressive disorder.

[0273] It is a still yet another object of the present invention toprovide pharmaceutical compositions for use in therapeutic methods whichcomprise or are based upon the PRODH, its subunits, their bindingpartner(s), as well as molecules whose activity or production depends onPRODH; or upon molecules or agents or drugs that control the production,stability and degradation, or that mimic the activities of the PRODH.

[0274] It is yet still another object of the present invention toprovide methods of determining a susceptibility for, or presence of,schizophrenia, or a disease or disorder related thereto, by determiningthe levels of an enzyme involved in proline catabolism, wherein such anenzyme comprises PRODH.

[0275] It is yet still another object of the present invention toprovide numerous methods for the selection of a drug or therapeuticagent to treat potentially schizophrenia, or a disease or disorderrelated thereto, such as obsessive compulsive disorder (OCD), bipolardisorder (BP) or major depressive disorder.

[0276] It is yet still another object of the invention to provide theDNA sequence of the wild-type murine Prodh gene, and an isolated variantof the allele of the wild-type murine Prodh gene.

[0277] It is yet still another object of the invention to provide theamino acid sequence of wild-type murine Prodh protein, as well as amutant murine Prodh protein.

[0278] It is yet still another object of the invention to providemammalian assays for determining whether a drug or agent has the abilityto treat schizophrenia or a disease or disorder related thereto. Suchassays involve Pro/Re mice described in the Example, and the PPI test,described below.

[0279] These and other aspects of the present invention will be betterappreciated by reference to the following drawings and DetailedDescription.

BRIEF DESCRIPTION OF THE DRAWINGS

[0280]FIG. 1: (A) Multiple alignment of the predicted protein sequencesof H. sapiens, M. musculus, D. melanogaster and S. cerevisiae prolinedehydrogenase. Shaded areas highlight identities. Also, an asterisk (Q)indicates a mutation in murine Prodh. (B) Localization of the humanproline dehydrogenase gene to chromosome 22q11: Southern blothybridization analysis of an array of nine PACs mapped and orderedwithin the 22q11 microdeletion [Carlson et al., 1997]. A human PRODHcDNA fragment was used as a hybridization probe. Only PAC-P457M14 (Q)provided a positive signal. The positions of the COMT and DGCR6 genes,previously mapped in this region [M. H. Grossman, B. S. Emanuel, M. L.Budarf, Genomics 12. 822 (1992); S. Demczuk, G. Thomas, A. Aurias, Hum.Mol. Genet. 5, 633 (1996)] are indicated as reference points. MarkersD22S427 and D22S264, that flank the smallest 22q11 deletion associatedwith schizophrenia as presented in [Karayiorgou et al., 1995], are alsoindicated. (C) High resolution Northern blot analysis of the brainexpression pattern of human PRODH (human brain mRNA filter was purchasedfrom Clontech (Palo Alto, Calif.)); hybridization to a human β-actinprobe was used to confirm equal loading of undegraded mRNA in each lane.

[0281]FIG. 2: (A) Northern blot analysis of expression of the mouseProdh gene. The size of the markers in kilobases are indicated to theleft of the blot. (B) Mutational analysis of the mouse Prodh gene fromthe Pro/Re strain: segment of antisense genomic sequence including theG—>T (C—>A) substitution (arrow) identified 135 base pairs upstream ofthe native termination codon, introducing a premature translationaltermination. (C) Analysis of plasma proline and glutamate levels inhomozygous and wild type F3 generation mice from a cross between theoriginal Pro/Re and C57/B6 wild type strain. The presence of theidentified mutation correlates with increased levels of proline in F3mice (p<0.0001). Y-axis values represent mmoles/L (±SEM). (D) Basallevels of glutamate, GABA and aspartate in the brains of Prodh-deficientmice and littermate controls. Neurotransmitter levels were analyzed oneweek after the termination of the behavioral analysis. Panels show basallevels of glutamate, GABA and aspartate in the frontal cortex,hypothalamus, amygdala, and hippocampus of homozygous and wild typeanimals of both sexes. Y-axis values are mean (±S.E.M). GAG/GAG wildtype mice (gray bar); TAG/TAG homozygous mice (solid bar). Data wasanalyzed by two-way ANOVA (*p<0.05, ****p<0.0001).

[0282]FIG. 3: Sensorimotor gating in Prodh-deficient mice: (A) Schematicoutline of the experiment. (B) Prepulse inhibition of an acousticstartle response; prepulse inhibition was examined for a combination oftwo startle dB levels (110 dB and 115 dB) and two prepulse dB levels (82dB and 90 dB); higher Y-axis values (mean ±SEM) represent greaterpercent inhibition. ANOVA with repeated measures revealed a significantattenuation in the overall level of PPI in the homozygous mutant micecompared to wild type littermates [F(1,66)=6.14, p=0.015]. (C) Amplitudeof startle response at two different startling stimuli; Y-axis values(mean ±SEM) represent weight-corrected peak amplitude startle (Vmax). Nosignificant differences were observed between genotypes at either 100 dB(p=0.0799) or 115 dB (p=0.125). (D) Habituation of the startle responseto repeated presentations of an 115 dB burst. Weight-corrected startlemagnitude values (Vmax) were averaged, expressed as a percentage of thefirst block and analyzed using ANOVA with repeated measures. Nosignificant differences were observed between genotypes (p=0.675).

[0283]FIG. 4: Locomotion and anxiety-like behaviors (collectively termedanxiety, reactivity or emotionality) of Prodh-deficient mice: (A) Openfield locomotion assay: No significant differences between genotypeswere observed in total distance traveled (p=0.747), stereotypic behavior(p=0.839), or time spent at the margin of the field (an estimate ofanxiety, p=0.078). Data were collected every 5 minutes over a 15 minuteperiod and analyzed with ANOVA with repeated measures. Because animalswere not preexposed to the chamber prior to testing, data collectedevery five minutes were also analyzed separately using two-way ANOVA(not shown) but no genotype or sex effect were observed. (B) Dark-lightassay: no significant differences were observed between genotypes inlatency to emerge into the lit compartment (p=0.597), or in horizontalactivity in either the lit (p=0.194) or the dark compartment (p=0.711).

[0284]FIG. 5: shows the results of the testing of families withschizophrenia to determine whether the polymorphism of the PRODH genecomprising a variation in SEQ ID NO:1, wherein the variation comprises asilent T to C transition at the first position of codon 497, whichintroduces a PuvII site is preferentially transmitted in such families.In particular, results of a Transmission Disequilibrium Test (TDT) aredisclosed which show the preferential transmission of a variant alleleof the PRODH gene comprising a DNA sequence having a variation in SEQ IDNO:1, wherein the variation comprises a silent T to C transition at thefirst position of codon 497.

[0285]FIG. 6 shows the human PRODH cDNA sequence (SEQ ID NO:1).Underlined nucleotides 1-3 make up the first codon of the sequence, andunderlined nucleotides 1549-1551 make up the termination codon.

[0286]FIG. 7 shows the amino acid sequence of human prolinedehydrogenase (SEQ ID NO:2).

[0287]FIG. 8 shows the murine Prodh cDNA sequence (SEQ ID NO:3).

[0288]FIG. 9 shows the amino acid sequence of murine prolinedehydrogenase (SEQ ID NO:4).

[0289]FIG. 10 shows the DNA sequence of a variant allele of the murineProdh gene (SEQ ID NO:7).

[0290]FIG. 11 shows the amino acid sequence of mutant murine Prodhprotein (SEQ ID NO:8).

[0291]FIG. 12 shows the DNA sequence of an isolated nucleic acidmolecule of the invention which encodes human proline dehydrogenase (SEQID NO:9). Underlined nucleotides 447-449 make up the first codon, andunderlined nucleotides 1995-1997 make up the termination codon.

DETAILED DESCRIPTION OF THE INVENTION

[0292] As explained above, the present invention is based upon thediscovery of the DNA sequences of human PRODH and murine Prodh, aheretofore unknown variant of murine Prodh which encodes a mutant Prodhprotein, and heretofore unknown variant alleles of the PRODH gene, thatencode heretofore unknown variant human proline dehydrogenase (PRODH)proteins.

[0293] In addition, the present invention is based upon Applicants'discovery that unexpectedly, a correlation exists between the presenceof schizophrenic symptoms in a subject, and the presence of a variantallele of the PRODH gene in the subject's genome. Hence, a variantallele of human PRODH can serve as a genetic marker to determine asusceptibility to, or presence of schizophrenia or a disease or disorderrelated thereto, such as obsessive compulsive disorder (OCD), bipolardisorder (BP) or major depressive disorder (MDD) in a subject.

[0294] Furthermore, the present invention extends to diagnostic methodsto determine a subject's increased or decreased susceptibility toschizophrenia or a disease or disorder related thereto. With the resultsof such methods, targeted prevention methods, early therapeuticintervention, and improved chronic treatment for schizophrenia or adisease or disorder related thereto are set forth herein and encompassedby the present invention. In addition, attending medical professionalsarmed with the results of such diagnostic methods can determine anappropriate regimen to treat the subject.

[0295] What's more, the present invention extends to assays to determinethe ability of a drug or agent can be used to treat schizophrenia or adisease or disorder related thereto.

[0296] Numerous terms and phrases are used throughout the instantApplication and are defined below:

[0297] As used herein “PRODH” refers to a wild type human prolinedehydrogenase gene, and “PRODH” refers to a wild-type human prolinedehydrogenase protein. An example of the PRODH gene comprises a DNAsequence of SEQ ID NO:1. Another example comprises the DNA sequence ofSEQ ID NO:9.

[0298] As used herein, the phrase “isolated variant allele of a humanproline dehydrogenase (PRODH) gene” refers to an assembly of nucleotidesthat includes cDNA and genomic DNA nucleic acids, which is a mutationalstate of the wild-type PRODH gene.

[0299] As used herein, an “isolated human variant proline dehydrogenase”or a “variant PRODH” refer to a protein which is a mutational state ofthe wild type proline PRODH.

[0300] As used herein, “Prodh” refers to a wild-type murine prolinedehydrogenase gene, and “Prodh” refers to a wild-type murine prolinedehydrogenase protein.

[0301] As used herein, the phrase “isolated variant allele of murineProdh gene” refers to an assembly of nucleotides that includes cDNA andgenomic DNA nucleic acids, which is a mutational state of the wild-typeProdh gene. A particular example of an isolated variant allele of themurine Prodh gene comprises a DNA sequence of FIG. 10 (SEQ ID NO:7).

[0302] As used herein, the phrase “mutant Prodh protein” or “variantProdh” refers to a protein encoded by an isolated variant allele ofmurine Prodh gene, which has an amino acid sequence that is differentfrom the amino acid sequence of wild-type murine Prodh protein. Aparticular example of wild-type murine Prodh protein comprises an aminoacid sequence of FIG. 9 (SEQ ID NO:4), while a mutant murine Prodhprotein comprises an amino acid sequence of FIG. 11 (SEQ ID NO:8). Acomparison of these two sequences shows that the mutant murine Prodh hasan amino acid sequence different from that of Prodh.

[0303] As used herein, the term “transition” refers to a mutationalevent in which one purine is replaced by another, or one pyrimidine isreplaced by another.

[0304] As used herein, the term “codon” refers to a triplet of bases ina DNA or RNA molecule that specifies or encodes the information for asingle amino acid.

[0305] As used herein, the phrase “F3 generation mouse from a cross ofPro/Re X C57B1/6J wild-type, wherein the F3 generation mouse has twocopies within its genome of an isolated variant allele of a Prodh genecomprising a DNA sequence of SEQ ID NO:7 which are capable of expressinga mutant Prodh comprising an amino acid sequence of SEQ ID NO:8” refersto the third generation mouse of a line of mice formed from a crossbetween the Pro/Re strain of mice and the wild-type C57B1/6J wild-typestrain, wherein the mouse is homozygous for the variant allele of Prodhcomprising the DNA sequence of SEQ ID NO:7, and expresses the variantProdh comprising a DNA sequence of SEQ ID NO:8.

[0306] As used herein, the phrase “F3 generation mouse from a cross ofPro/Re X C57B1/6J wild-type, wherein the F3 generation mouse has twocopies within its genome of an isolated Prodh gene comprising a DNAsequence of SEQ ID NO:3 which are capable of expressing a Prodhcomprising an amino acid sequence of SEQ ID NO:4” refers to the thirdgeneration mouse of a line of mice formed from a cross between thePro/Re strain of mice and the wild-type C57BL/6J wild-type strain,wherein the mouse is homozygous for the Prodh gene comprising the DNAsequence of SEQ ID NO:3, and expresses Prodh comprising a DNA sequenceof SEQ ID NO:4.

[0307] As used herein, the terms “schizophrenia”, “obsessive compulsivedisorder”, “bipolar disorder”, and “major depressive disorder” refer topsychiatric diseases or disorders that are readily understood by theskilled artisan and are set forth in American Psychiatric Associate(1994); Diagnostic and Statistical Manual of Mental Disorders, 4^(th)Edition. Washington, D.C.

[0308] As used herein, the term “susceptibility” to schizophrenia or adisease or disorder related thereto refers to a subject's potential ofbeing affected with such a disease or disorder.

[0309] As used herein, the terms “standard” and “control” refer to abodily sample, cell extract, etc. established for use as a rule or basisof comparison in measuring levels of activity of PRODH, results of PPI,etc.

[0310] As used herein, the term “combination” referring to variations inthe wild-type sequence, either amino acid residues or nucleotides,indicates that two or more of the discovered variations in theparticular DNA sequence of amino acid sequence can be present in variantallele of the wild-type nucleic acid molecule or protein.

[0311] An initial aspect of the invention extends to an isolated nucleicacid molecule comprising a DNA sequence of SEQ ID NO:1, degeneratevariants thereof, fragments thereof, or analogs or derivatives thereof.

[0312] Another aspect of the invention extends to an isolated nucleicacid molecule comprising a DNA sequence of SEQ ID NO:3, degeneratevariants thereof, fragments thereof, or analogs or derivatives thereof.

[0313] Another aspect of the present invention extends an isolatedvariant allele of the PRODH gene, wherein the PRODH gene comprises a DNAsequence of SEQ ID NO:1 or SEQ ID NO:9, and an isolated variant alleleof the PRODH comprises a DNA sequence having at least one variation inSEQ ID NO:1 or SEQ ID NO:9, wherein the at least one variationcomprises:

[0314] a G to A transition in the third position of codon 83;

[0315] a C to T transition in the first position of codon 101;

[0316] a G to A transition in the second position of codon 101;

[0317] a C to T transition in the first position of codon 247;

[0318] a C to T transition in the third position of codon 342;

[0319] a C to T transition in the third position of codon 421;

[0320] an A to G transition in the second position of codon 437;

[0321] a T to C transition in the first position of codon 497; or

[0322] a combination thereof.

[0323] Furthermore, the present invention is based on discovery thatsurprisingly and unexpectedly, a particular variant allele of the PRODHgene is present in a statistically significantly higher frequency insubjects suffering from a psychiatric disease or disorder.

[0324] Further explanation of this aspect of the invention is set forthinfra.

[0325] Consequently, an initial aspect of the present inventioncontemplates isolation of PRODH, Prodh, and heretofore unknown variantalleles of the human PRODH gene. As used herein, the term “gene” refersto an assembly of nucleotides that encode a polypeptide, and includescDNA and genomic DNA nucleic acids.

[0326] Furthermore, in accordance with the present invention there maybe employed conventional molecular biology, microbiology, andrecombinant DNA techniques within the skill of the art. Such techniquesare explained fully in the literature. See, e.g., Sambrook, Fritsch &Maniatis, Molecular Cloning: A Laboratory Manual, Second Edition (1989)Cold Spring Harbor Laboratory Press, Cold Spring Harbor, N.Y. (herein“Sambrook et al., 1989”); DNA Cloning: A Practical Approach, Volumes Iand II (D. N. Glover ed. 1985); Oligonucleotide Synthesis (M. J. Gaited. 1984); Nucleic Acid Hybridization [B. D. Hames & S. J. Higgins eds.(1985)]; Transcription And Translation [B. D. Hames & S. J. Higgins.

[0327] eds. (1984)]; Animal Cell Culture [R. I. Freshney, ed. (1986)];Immobilized Cells And Enzymes [IRL Press, (1986)]; B. Perbal, APractical Guide To Molecular Cloning (1984); F. M. Ausubel et al.(eds.), Current Protocols in Molecular Biology, John Wiley & Sons, Inc.(1994).

[0328] Therefore, if appearing herein, the following terms shall havethe definitions set out below.

[0329] A “vector” is a replicon, such as plasmid, phage or cosmid, towhich another DNA segment may be attached so as to bring about thereplication of the attached segment. A “replicon” is any genetic element(e.g., plasmid, chromosome, virus) that functions as an autonomous unitof DNA replication in vivo, i.e., capable of replication under its owncontrol.

[0330] A “cassette” refers to a segment of DNA that can be inserted intoa vector at specific restriction sites. The segment of DNA encodes apolypeptide of interest, and the cassette and restriction sites aredesigned to ensure insertion of the cassette in the proper reading framefor transcription and translation.

[0331] “Heterologous” DNA refers to DNA not naturally located in thecell, or in a chromosomal site of the cell. Preferably, the heterologousDNA includes a gene foreign to the cell.

[0332] As used herein, the term “wild-type” refers to the most commonlyobserved phenotype or genotype, designated as the norm.

[0333] A “nucleic acid molecule” refers to the phosphate ester polymericform of ribonucleosides (adenosine, guanosine, uridine or cytidine; “RNAmolecules”) or deoxyribonucleosides (deoxyadenosine, deoxyguanosine,deoxythymidine, or deoxycytidine; “DNA molecules”), or any phosphoesteranalogs thereof, such as phosphorothioates and thioesters, in eithersingle stranded form, or a double-stranded form comprising coding andcomplementary strands. Double stranded DNA-DNA, DNA-RNA and RNA-RNAhelices are possible. The term nucleic acid molecule, and in particularDNA or RNA molecule, refers only to the primary and secondary structureof the molecule, and does not limit it to any particular tertiary forms.Thus, this term includes double-stranded DNA found, inter alia, inlinear or circular DNA molecules (e.g., restriction fragments),plasmids, and chromosomes. In discussing the structure of particulardouble-stranded DNA molecules, sequences may be described hereinaccording to the normal convention of giving only the sequence in the 5′to 3′ direction along the nontranscribed strand of DNA (i.e., the strandhaving a sequence homologous to the mRNA). A “recombinant DNA molecule”is a DNA molecule that has undergone a molecular biologicalmanipulation.

[0334] A nucleic acid molecule is “hybridizable” to another nucleic acidmolecule, such as a cDNA, genomic DNA, or RNA, when a single strandedform of the nucleic acid molecule, e.g. either the coding strand or thestrand complementary to the coding strand, can anneal to the othernucleic acid molecule under the appropriate conditions of temperatureand solution ionic strength (see Sambrook et al., supra). The conditionsof temperature and ionic strength determine the “stringency” of thehybridization. For preliminary screening for homologous nucleic acids,low stringency hybridization conditions, corresponding to a T_(m) of50°, can be used, e.g., 5× SSC, 0.1% SDS, 0.25% milk, and no formamide;or 30% formamide, 5× SSC, 0.5% SDS). Moderate stringency hybridizationconditions correspond to a higher T_(m), e.g., a T_(m) of 55° C., 40%formamide, with 5× or 6× SSC. High stringency hybridization conditionscorrespond to the highest T_(m), e.g., a T_(m) of 60-65° C., 50%formamide, 5× or 6× SSC. Hybridization requires that the two nucleicacids contain complementary sequences, although depending on thestringency of the hybridization, mismatches between bases are possible.The appropriate stringency for hybridizing nucleic acids depends on thelength of the nucleic acids and the degree of complementation, variableswell known in the art. The greater the degree of similarity or homologybetween two nucleotide sequences, the greater the value of T_(m) forhybrids of nucleic acids having those sequences. The relative stability(corresponding to higher T_(m)) of nucleic acid hybridizations decreasesin the following order: RNA:RNA, DNA:RNA, DNA:DNA. For hybrids ofgreater than 100 nucleotides in length, equations for calculating T_(m)have been derived (see Sambrook et al., supra, 9.50-9.51). Forhybridization with shorter nucleic acids, i.e., oligonucleotides, theposition of mismatches becomes more important, and the length of theoligonucleotide determines its specificity (see Sambrook et al., supra,11.7-11.8). Preferably a minimum length for a hybridizable nucleic acidis at least about 10 nucleotides; particularly at least about 15nucleotides; more particularly at least about 20 nucleotides; even moreparticularly at least about 30 nucleotides, and yet more particularly atleast about 40 nucleotides, and most particularly about 50 nucleotides.

[0335] In a specific embodiment, the term “standard hybridizationconditions” refers to a T_(m) of 55° C., and utilizes conditions as setforth above. In a preferred embodiment, the T_(m) is 60° C.; in a morepreferred embodiment, the T_(m) is 65° C.

[0336] “Homologous recombination” refers to the insertion of a foreignDNA sequence of a vector in a chromosome. Preferably, the vector targetsa specific chromosomal site for homologous recombination. For specifichomologous recombination, the vector will contain sufficiently longregions of homology to sequences of the chromosome to allowcomplementary binding and incorporation of the vector into thechromosome. Longer regions of homology, and greater degrees of sequencesimilarity, may increase the efficiency of homologous recombination.

[0337] A DNA “coding sequence” is a double-stranded DNA sequence whichis transcribed and translated into a polypeptide in a cell in vitro orin vivo when placed under the control of appropriate regulatorysequences. The boundaries of the coding sequence are determined by astart codon at the 5′ (amino) terminus and a translation stop codon atthe 3′ (carboxyl) terminus. A coding sequence can include, but is notlimited to, prokaryotic sequences, cDNA from eukaryotic mRNA, genomicDNA sequences from eukaryotic (e.g., mammalian) DNA, and even syntheticDNA sequences. If the coding sequence is intended for expression in aeukaryotic cell, a polyadenylation signal and transcription terminationsequence will usually be located 3′ to the coding sequence.

[0338] Transcriptional and translational control sequences are DNAregulatory sequences, such as promoters, enhancers, terminators, and thelike, that provide for the expression of a coding sequence in a hostcell. In eukaryotic cells, polyadenylation signals are controlsequences.

[0339] A “promoter sequence” or “promoter” is a DNA regulatory regioncapable of binding RNA polymerase in a cell and initiating transcriptionof a downstream (3′ direction) coding sequence. For purposes of definingthe present invention, the promoter sequence is bounded at its 3′terminus by the transcription initiation site and extends upstream (5′direction) to include the minimum number of bases or elements necessaryto initiate transcription at levels detectable above background. Withinthe promoter sequence will be found a transcription initiation site(conveniently defined for example, by mapping with nuclease S1), as wellas protein binding domains (consensus sequences) responsible for thebinding of RNA polymerase.

[0340] A coding sequence is “under the control” of transcriptional andtranslational control sequences in a cell when RNA polymerasetranscribes the coding sequence into mRNA, which is then trans-RNAspliced and translated into the protein encoded by the coding sequence.

[0341] A coding sequence is “operatively associated with” atranscriptional and translational control sequences, such as a promoterfor example, when RNA polymerase transcribes the coding sequence intomRNA, which in turn is translated into a protein encoded by the codingsequence.

[0342] A “signal sequence” is included at the beginning of the codingsequence of a protein to be expressed on the surface of a cell. Thissequence encodes a signal peptide, N-terminal to the mature polypeptide,that directs the host cell to translocate the polypeptide. The term“translocation signal sequence” is used herein to refer to this sort ofsignal sequence. Translocation signal sequences can be found associatedwith a variety of proteins native to eukaryotes and prokaryotes, and areoften functional in both types of organisms.

[0343] An “expression control sequence” is a DNA sequence that controlsand regulates the transcription and translation of another DNA sequence.A coding sequence is “under the control” of transcriptional andtranslational control sequences in a cell when RNA polymerasetranscribes the coding sequence into mRNA, which is then translated intothe protein encoded by the coding sequence.

[0344] The term “oligonucleotide” as used herein in referring to theprobe of the present invention, is defined as a molecule comprised oftwo or more ribonucleotides, preferably more than three. Its exact sizewill depend upon many factors which, in turn, depend upon the ultimatefunction and use of the oligonucleotide.

[0345] The term “primer” as used herein refers to an oligonucleotide,whether occurring naturally as in a purified restriction digest orproduced synthetically, which is capable of acting as a point ofinitiation of synthesis when placed under conditions in which synthesisof a primer extension product, which is complementary to a nucleic acidstrand, is induced, i.e., in the presence of nucleotides and an inducingagent such as a DNA polymerase and at a suitable temperature and pH. Theprimer may be either single-stranded or double-stranded and must besufficiently long to prime the synthesis of the desired extensionproduct in the presence of the inducing agent. The exact length of theprimer will depend upon many factors, including temperature, source ofprimer and use of the method. For example, for diagnostic applications,depending on the complexity of the target sequence, the oligonucleotideprimer typically contains 15-50 or more nucleotides, although it maycontain fewer nucleotides.

[0346] The primers herein are selected to be “substantially”complementary to different strands of a particular target DNA sequence.This means that the primers must be sufficiently complementary tohybridize with their respective strands. Therefore, the primer sequenceneed not reflect the exact sequence of the template. For example, anon-complementary nucleotide fragment may be attached to the 5′ end ofthe primer, with the remainder of the primer sequence beingcomplementary to the strand. Alternatively, non-complementary bases orlonger sequences can be interspersed into the primer, provided that theprimer sequence has sufficient complementary with the sequence of thestrand to hybridize therewith and thereby form the template for thesynthesis of the extension product.

[0347] A cell has been “transfected” by exogenous or heterologous DNAwhen such DNA has been introduced inside the cell. A cell has been“transformed” by exogenous or heterologous DNA when the transfected DNAeffects a phenotypic change. Preferably, the transforming DNA should beintegrated (covalently linked) into chromosomal DNA making up the genomeof the cell.

[0348] A “clone” is a population of cells derived from a single cell orcommon ancestor by mitosis. A “cell line” is a clone of a primary cellthat is capable of stable growth in vitro for many generations.

[0349] As used herein, the phrase “an isolated nucleic acid molecule ofthe invention” refers to any of the following:

[0350] a) an isolated nucleic acid molecule comprising a DNA sequence ofSEQ ID NO:1, degenerate variants thereof, fragments thereof, or analogsor derivatives thereof;

[0351] b) an isolated nucleic acid hybridizable under standardhybridization conditions to an isolated nucleic acid molecule comprisinga DNA sequence of SEQ ID NO:1, degenerate variants thereof, fragmentsthereof, or analogs or derivatives thereof;

[0352] c) an isolated nucleic acid molecule comprising a DNA sequence ofSEQ ID NO:3, degenerate variants thereof, fragments thereof, or analogsor derivatives thereof;

[0353] d) an isolated nucleic acid molecule hybridizable under standardhybridization conditions to an isolated nucleic acid molecule comprisinga DNA sequence of SEQ ID NO:3, degenerate variants thereof, fragmentsthereof, or analogs or derivatives thereof,

[0354] e) an isolated variant allele of a PRODH gene, wherein theisolated variant allele comprises a DNA sequence having at least onevariation in SEQ ID NO:1, wherein the at least one variation comprises:

[0355] a G to A transition in the third position of codon 83;

[0356] a C to T transition in the first position of codon 101;

[0357] a G to A transition in the second position of codon 101;

[0358] a C to T transition in the first position of codon 247;

[0359] a C to T transition in the third position of codon 342;

[0360] a C to T transition in the third position of codon 421;

[0361] an A to G transition in the second position of codon 437;

[0362] a T to C transition in the first position of codon 497; or

[0363] a combination thereof along with degenerate variants thereof,fragments thereof, or analogs or derivatives thereof;

[0364] f) an isolated nucleic acid molecule hybridizable under standardhybridization conditions to an isolated variant allele of the PRODHgene, degenerate variants thereof, fragments thereof, or analogs orderivatives thereof;

[0365] g) an isolated variant of a murine Prodh gene comprising a DNAsequence of SEQ ID NO:7, degenerate variants thereof, fragments thereof,or analogs or derivatives thereof;

[0366] h) an isolated nucleic acid molecule hybridizable under standardhybridization conditions to an isolated nucleic acid molecule comprisingthe DNA sequence of SEQ ID NO:7, degenerate variants thereof, fragmentsthereof, or analogs or derivatives thereof;

[0367] i) an isolated nucleic acid molecule comprising a DNA sequence ofSEQ ID NO:9, degenerate variants thereof, fragments thereof, or analogsor derivatives thereof; and

[0368] j) an isolated nucleic acid molecule hybridizable under standardhybridization conditions to an isolated nucleic acid molecule comprisinga DNA sequence of SEQ ID NO:9, degenerate variants thereof, fragmentsthereof, or analogs or derivatives thereof.

[0369] As used herein, an “isolated protein of the invention” refers toany of the following:

[0370] a) an isolated protein comprising an amino acid sequence of SEQID NO:2, conservative variants thereof, fragments thereof, or analogs orderivatives thereof;

[0371] b) an isolated protein comprising an amino sequence of SEQ IDNO:4, conservative variants thereof, fragments thereof, or analogs orderivatives thereof; and

[0372] c) an isolated protein comprising an amino acid sequence havingat least one variation in SEQ ID NO:2, wherein the at least onevariation comprises:

[0373] Arg101Trp;

[0374] Arg101Glu;

[0375] Glu437Arg; or

[0376] a combination thereof;

[0377] d) an isolated mutant variant proline dehydrogenase (Prodh)comprising an amino acid sequence of SEQ ID NO:8, conservative variantsthereof, fragments thereof, or analogs or derivatives thereof.

[0378] Degenerate Variants of an Isolated Nucleic Acid Molecule of theInvention, and Conservative Variants of an Isolated Protein of theInvention

[0379] Due to the degenerate nature of codons in the genetic code, anisolated protein of the invention can be encoded by nucleic acidmolecules other than an isolated nucleic acid molecule of the invention.“Degenerate nature” refers to the use of different three-letter codonsto specify a particular amino acid pursuant to the genetic code. It iswell known in the art that the following codons can be usedinterchangeably to code for each specific amino acid:

[0380] Phenylalanine (Phe or F)

[0381] UUU or UUC

[0382] Leucine (Leu or L)

[0383] UUA or UUG or CUU or CUC or CUA or CUG

[0384] Isoleucine (IIe or I)

[0385] AUU or AUC or AUA

[0386] Methionine (Met or M)

[0387] AUG

[0388] Valine (Val or V)

[0389] GUU or GUC of GUA or GUG

[0390] Serine (Ser or S)

[0391] UCU or UCC or UCA or UCG or AGU or AGC

[0392] Proline (Pro or P)

[0393] CCU or CCC or CCA or CCG

[0394] Threonine (Thr or T)

[0395] ACU or ACC or ACA or ACG

[0396] Alanine (Ala or A)

[0397] GCU or GCG or GCA or GCG

[0398] Tyrosine (Tyr or Y)

[0399] UAU or UAC

[0400] Histidine (His or H)

[0401] CAU or CAC

[0402] Glutamine (Gln or Q)

[0403] CAA or CAG

[0404] Asparagine (Asn or N)

[0405] AAU or AAC

[0406] Lysine (Lys or K)

[0407] AAA or AAG

[0408] Aspartic Acid (Asp or D)

[0409] GAU or GAC

[0410] Glutamic Acid (Glu or E)

[0411] GAA or GAG

[0412] Cysteine (Cys or C)

[0413] UGU or UGC

[0414] Arginine (Arg or R)

[0415] CGU or CGC or CGA or CGG or AGA or AGG

[0416] Glycine (Gly or G)

[0417] GGU or GGC or GGA or GGG

[0418] Tryptophan (Trp or W)

[0419] UGG

[0420] Termination codon

[0421] UAA (ochre) or UAG (amber) or UGA (opal)

[0422] It should be understood that the codons specified above are forRNA sequences. The corresponding codons for DNA have a T substituted forU.

[0423] Likewise, conservative isolated variants of an PRODH protein ofthe present invention include, but are not limited to, those containing,as a primary amino acid sequence, substitutions of amino acid residuesin the amino acid sequences of SEQ ID NO:2, as well as in a variantPRODH protein of the invention having an amino acid sequence asdescribed above, a murine Prodh comprising an amino acid sequence of SEQID NO:4, and a mutant murine Prodh comprising an amino acid sequence ofSEQ ID NO:8, wherein the substituted amino acids are functionallyequivalent to the amino acid residues for which they substitute. Forexample, one or more amino acid residues within the sequence can besubstituted by another amino acid of a similar polarity, which acts as afunctional equivalent, resulting in a silent alteration. Substitutes foran amino acid within the sequence may be selected from other members ofthe class to which the amino acid residue belongs. For example, thenonpolar (hydrophobic) amino acids include alanine, leucine, isoleucine,valine, proline, phenylalanine, tryptophan and methionine. Amino acidscontaining aromatic ring structures are phenylalanine, tryptophan, andtyrosine. The polar neutral amino acids include glycine, serine,threonine, cysteine, tyrosine, asparagine, and glutamine. The positivelycharged (basic) amino acids include arginine, lysine and histidine. Thenegatively charged (acidic) amino acids include aspartic acid andglutamic acid. Such alterations will not be expected to affect apparentmolecular weight as determined by polyacrylamide gel electrophoresis, orisoelectric point.

[0424] Particularly preferred substitutions are:

[0425] Lys for Arg and vice versa such that a positive charge may bemaintained;

[0426] Glu for Asp and vice versa such that a negative charge may bemaintained;

[0427] Ser for Thr such that a free —OH can be maintained; and

[0428] Gln for Asn such that a free NH₂ can be maintained.

[0429] Amino acid substitutions may also be introduced to substitute anamino acid with a particularly preferable property. For example, a Cysmay be introduced at a potential site for disulfide bridges with anotherCys. A His may be introduced as a particularly “catalytic” site (i.e.,His can act as an acid or base and is the most common amino acid inbiochemical catalysis). Pro may be introduced because of itsparticularly planar structure, which induces β-turns in the protein'sstructure. As a result of such substitutions, “conservative variants” ofan amino acid sequence of the invention are formed.

[0430] Fragments of Isolated Nucleic Acid Molecules and IsolatedProteins of the Invention

[0431] Further, as used herein, a “fragment” of an isolated nucleic acidmolecule of the invention is defined as an isolated nucleic acidmolecule comprising at least 10 contiguous nucleotides, particularly atleast 20 contiguous nucleotides, more particularly at least 30contiguous nucleotides, and even more particularly at least 40contiguous nucleotides of an isolated nucleic acid molecule of theinvention in the same 5′-3′ order the contiguous nucleotides appear inthe isolated nucleic acid molecule of the invention. Fragments of anisolated nucleic acid molecule of the invention can readily be preparedby digesting an isolated nucleic acid molecule of the invention with arestriction endonuclease. Alternatively, one may use DNAse in thepresence of manganese to fragment the DNA, or the DNA can be physicallysheared, as for example, by sonication. The DNA fragments of an isolatednucleic acid molecule of the invention can then be separated accordingto size by standard techniques, including but not limited to, agaroseand polyacrylamide gel electrophoresis and column chromatography. Oncefragments of the isolated nucleic acid molecule have themselves beenseparated, they can be readily inserted the fragments into expressionvectors. As a result, the peptides and polypeptides, which arethemselves fragments of an isolated protein of the invention can readilybe produced by one of ordinary skill in the art.

[0432] Likewise, a “fragment” of protein of the invention comprises atleast 10 contiguous amino acid residues, particularly at least 15contiguous amino acid residues, even more particularly at least 20contiguous amino acid residues, and most particularly at least 25contiguous amino acid residues of a protein of the invention, in the Nterminus to C terminus order in which the contiguous residues occur inthe protein of the invention. One of ordinary skill in the art canreadily prepare such fragments using recombinant DNA techniques withfragments of isolated nucleic acid molecules of the invention, bydigesting a protein of the invention with a protease, chemicallycleaving a protein of the invention with chemical reagents such as CNBr,or synthesizing the fragment using routine solid support methods astaught by Merrifield.

[0433] As used herein, the term “sequence homology” in all itsgrammatical forms refers to the relationship between proteins thatpossess a “common evolutionary origin,” including proteins fromsuperfamilies (e.g., the immunoglobulin superfamily) and homologousproteins from different species (e.g., myosin light chain, etc.) (Reecket al., 1987, Cell 50:667).

[0434] Accordingly, the term “sequence similarity” in all itsgrammatical forms refers to the degree of identity or correspondencebetween nucleic acid or amino acid sequences of proteins that do notshare a common evolutionary origin (see Reeck et al., supra). However,in common usage and in the instant application, the term “homologous,”when modified with an adverb such as “highly,” may refer to sequencesimilarity and not a common evolutionary origin.

[0435] In a specific embodiment, two DNA sequences are “substantiallyhomologous” or “substantially similar” when at least about 50%(preferably at least about 75%, and most preferably at least about 90 or95%) of the nucleotides match over the defined length of the DNAsequences. Sequences that are substantially homologous can be identifiedby comparing the sequences using standard software available in sequencedata banks, or in a Southern hybridization experiment under, forexample, stringent conditions as defined for that particular system.Defining appropriate hybridization conditions is within the skill of theart. See, e.g., Maniatis et al., supra; DNA Cloning, Vols. I & II,supra; Nucleic Acid Hybridization, supra.

[0436] Similarly, in a particular embodiment, two amino acid sequencesare “substantially homologous” or “substantially similar” when greaterthan 30% of the amino acids are identical, or greater than about 60% aresimilar (functionally identical). Preferably, the similar or homologoussequences are identified by alignment using, for example, the GCG(Genetics Computer Group, Program Manual for the GCG Package, Version 7,Madison, Wis.) pileup program-using default parameters.

[0437] The term “corresponding to” is used herein to refer to similar orhomologous sequences, whether the exact position is identical ordifferent from the molecule to which the similarity or homology ismeasured. Thus, the term “corresponding to” refers to the sequencesimilarity, and not the numbering of the amino acid residues ornucleotide bases.

[0438] A variant allele of the human PRODH gene, whether genomic DNA orcDNA, can be isolated from any source, particularly from a human cDNA orgenomic library. Likewise, a variant allele of Prodh, whether genomic orcDNA can be isolated from any source, particularly from a murine cDNA orgenomic library. Methods for obtaining a variant allele of a PRODH geneor a Prodh gene are well known in the art, as described above (see,e.g., Sambrook et al., 1989, supra).

[0439] The DNA may be obtained by standard procedures known in the artfrom cloned DNA (e.g., a DNA “library”), and preferably is obtained froma cDNA library prepared from tissues with high level expression of aPRODH protein, an isolated variant thereof, or Prodh or an isolatedvariant thereof, by chemical synthesis, by cDNA cloning, or by thecloning of genomic DNA, or fragments thereof, purified from the desiredcell (See, for example, Sambrook et al., 1989, supra; Glover, D. M.(ed.), 1985, DNA Cloning: A Practical Approach, MRL Press, Ltd., Oxford,U.K. Vol. 1, II). Clones derived from genomic DNA may contain regulatoryand intron DNA regions in addition to coding regions; clones derivedfrom cDNA will not contain intron sequences. Whatever the source, anisolated nucleic acid molecule of the invention can be molecularlycloned into a suitable vector for propagation.

[0440] Once the DNA fragments are generated, identification of aspecific DNA fragment comprising an isolated nucleic acid molecule ofthe invention may be accomplished in a number of ways. For example, ifan amount of a portion of an isolated nucleic acid molecule is availableand can be purified and labeled, the generated DNA fragments may bescreened by nucleic acid hybridization to a labeled probe (Benton andDavis, 1977, Science 196:180; Grunstein and Hogness, 1975, Proc. Natl.Acad. Sci. U.S.A. 72:3961). For example, a set of oligonucleotidescorresponding to the partial amino acid sequence information obtainedSEQ ID NO: 2 or a variant human PRODH protein, SEQ ID NO:4, or SEQ IDNO:8 of the present invention can be prepared and used as probes for anisolated nucleic acid molecule of the invention, as was done in aspecific example, infra, or as primers for cDNA or mRNA (e.g., incombination with a poly-T primer for RT-PCR). Preferably, a fragment isselected that is highly unique to an isolated nucleic acid molecule ofthe invention. Those DNA fragments with substantial homology to theprobe will hybridize. As noted above, the greater the degree ofhomology, the more stringent hybridization conditions can be used.

[0441] Further selection can be carried out on the basis of theproperties the protein produced from expression of an isolated nucleicacid molecule of the invention. For example, an isolated variant alleleof a PRODH gene of the present invention can be isolated if it encodes avariant PRODH protein having an isoelectric, electrophoretic, amino acidcomposition, or partial amino acid sequence different from PRODHproduced from the expression of the PRODH gene (SEQ ID NO:1) herein.Thus, the presence of a variant allele of a PRODH gene of the presentinvention may be detected by assays based on the physical, chemical, orimmunological properties of its expressed product. For example, cDNAclones, or DNA clones which hybrid-select the proper mRNAs, can beselected which produce a protein that, e.g., has differentelectrophoretic migration, isoelectric focusing or non-equilibrium pHgel electrophoresis behavior, proteolytic digestion maps, or antigenicproperties as are known for a PRODH protein comprising an amino acidsequence of SEQ ID NO:2. Such selection can also be made between Prodhand an isolated variant of Prodh.

[0442] An isolated variant allele of a PRODH gene of the presentinvention, for example, can also be identified by mRNA selection, i.e.,by nucleic acid hybridization followed by in vitro translation. In thisprocedure, nucleotide fragments are used to isolate complementary mRNAsby hybridization. Such DNA fragments may represent available, purifiedDNA of an isolated variant allele of a human PRODH gene of the presentinvention, or may be synthetic oligonucleotides designed from thepartial amino acid sequence information.

[0443] Immunoprecipitation analysis or functional assays of the in vitrotranslation products of the products of the isolated mRNAs identifiesthe mRNA and, therefore, the complementary DNA fragments, that containthe desired sequences. Naturally, these techniques can also be used toidentify isolated variant of Prodh.

[0444] Furthermore, a detectably labeled isolated nucleic acid moleculeof the present invention can be prepared by one of ordinary skill in theart. Once detectably labeled, an isolated nucleic acid molecule of theinvention degenerate variants thereof, fragments thereof, or analogs orderivatives thereof, can then be used as a probe to identify homologousDNA fragments from among other genomic DNA fragments. Suitable labelsinclude enzymes, radioactive isotopes, fluorophores (e.g., fluoresceneisothiocyanate (FITC), phycoerythrin (PE), Texas red (TR), rhodamine,free or chelated lanthanide series salts, especially Eu³⁺, to name a fewfluorophores), chromophores, radioisotopes, chelating agents, dyes,colloidal gold, latex particles, ligands (e.g., biotin), andchemiluminescent agents. When a control marker is employed, the same ordifferent labels may be used for the receptor and control marker.

[0445] In the instance where a radioactive label, such as the isotopes³H, ¹⁴C, ³²P, ³⁵S, ³⁶Cl, ⁵¹Cr, ⁵⁷Co, ⁵⁸Co, ⁵⁹Fe, ⁹⁰Y, ¹²⁵I, ¹³¹I and¹⁸⁶Re are used, known currently available counting procedures may beutilized. In the instance where the label is an enzyme, detection may beaccomplished by any of the presently utilized colorimetric,spectrophotometric, filuorospectrophotometric, amperometric orgasometric techniques known in the art.

[0446] Direct labels are one example of labels which can be usedaccording to the present invention. A direct label has been defined asan entity, which in its natural state, is readily visible, either to thenaked eye, or with the aid of an optical filter and/or appliedstimulation, e.g. U. V. light to promote fluorescence. Examples ofcolored labels, which can be used according to the present inventioninclude metallic sol particles, for example, gold sol particles such asthose described by Leuvering (U.S. Pat. No. 4,313,734); dye solparticles such as described by Gribnau et al. (U.S. Pat. No. 4,373,932)and May et al. (WO 88108534); dyed latex such as described by May,supra, Snyder (EP-A 0 280 559 and 0 281 327); or dyes encapsulated inliposomes as described by Campbell et al. (U.S. Pat. No. 4,703,017).Other direct labels include a radionucleotide, a fluorescent moiety or aluminescent moiety. In addition to these direct labeling devices,indirect labels comprising enzymes can also be used according to thepresent invention. Various types of enzyme linked immunoassays are wellknown in the art, for example, alkaline phosphatase and horseradishperoxidase, lysozyme, glucose-6-phosphate dehydrogenase, lactatedehydrogenase, and urease. These and others have been discussed indetail by Eva Engvall in Enzyme Immunoassay ELISA and EMIT in Methods inEnzymology, 70. 419-439, 1980 and in U.S. Pat. No. 4,857,453.

[0447] Other labels for use in the invention include magnetic beads ormagnetic resonance imaging labels.

Cloning Vectors

[0448] As explained above, the present invention extends to variouscloning vectors. In particular, the present invention extends to acloning vector comprising an isolated nucleic acid molecule of theinvention and an origin of replication.

[0449] A large number of vector-host systems known in the art may beused to clone an isolated nucleic acid molecule of the invention.Possible vectors include, but are not limited to, plasmids or modifiedviruses. The vector system used however must be compatible with the hostcell. Examples of vectors that are commercially available and haveapplications herein include, but are not limited to, E. coli,bacteriophages such as lambda derivatives, or plasmids such as pBR322derivatives or pUC plasmid derivatives, e.g., pGEX vectors, pmal-c,pFLAG, etc. Furthermore, insertion of an isolated nucleic acid moleculeof the invention as described above into a cloning vector can be readilyaccomplished by one of ordinary skill in the art. For example, insertioncan be accomplished by ligating an isolated nucleic acid molecule of theinvention into a cloning vector which has complementary cohesivetermini. However, if the complementary restriction sites used tofragment the isolated nucleic acid molecule of the invention, to form acassette are not present in the cloning vector, the ends of the cassettemay be enzymatically modified using procedures well known to one ofordinary skill in the art. Once modified, the cassette can be readilyligated into a commercially available cloning vector. Alternatively, anysite desired may be produced by ligating nucleotide sequences (linkers)onto the DNA termini of the cassette; these ligated linkers may comprisespecific chemically synthesized oligonucleotides encoding restrictionendonuclease recognition sequences.

[0450] Once an isolated nucleic acid molecule of the invention isinserted into a vector, the vector can then be introduced into hostcells via transformation, transfection, infection, electroporation,etc., so that many copies of the isolated nucleic acid molecule of theinvention can be generated. Preferably, the cloned isolated variantallele or isolated nucleic acid molecule hybridizable thereto understandard hybridization conditions is contained on a shuttle vectorplasmid, which provides for expansion in a cloning cell, e.g., E. coli,and facile purification for subsequent insertion into an appropriateexpression cell line, if such is desired. For example, a shuttle vector,which is a vector that can replicate in more than one type of organism,can be prepared for replication in both E. coli and Saccharomycescerevisiae by linking sequences from an E. coli plasmid with sequencesfrom the yeast 2 μplasmid.

[0451] In an alternative method, an isolated nucleic acid molecule ofthe invention may be identified and isolated after insertion into asuitable cloning vector in a “shot gun” approach. Enrichment for anisolated variant allele, for example, by size fractionation, can be donebefore insertion into the cloning vector.

Expression Vectors

[0452] An isolated nucleic acid molecule of the inventioin can beinserted into an appropriate expression vector, i.e., a vector whichcontains the necessary elements for the transcription and translation ofthe inserted protein-coding sequence such that the protein-codingsequence is operatively associated with a promoter. A DNA sequence is“operatively associated” to an expression control sequence, such as apromoter, when the expression control sequence controls and regulatesthe transcription and translation of that DNA sequence. The term“operatively associated” includes having an appropriate start signal(e.g., ATG) in front of the DNA sequence to be expressed and maintainingthe correct reading frame to permit expression of the DNA sequence underthe control of the expression control sequence and production of thedesired product encoded by the DNA sequence. If an isolated nucleic acidmolecule of the invention does not contain an appropriate start signal,such a start signal can be readily inserted into the expression vectorin front of (5′ of) protein encoding nucleic acid molecule inserted intothe expression vector using methods readily understood and available toone of ordinary skill in the art.

[0453] Both cDNA and genomic sequences can be cloned and expressed undercontrol of such regulatory sequences. An expression vector alsopreferably includes a replication origin.

[0454] The necessary transcriptional and translational signals can beprovided on a recombinant expression vector, or they may be supplied byan isolated nucleic acid molecule of the invention inserted into anexpression vector.

[0455] Potential host-vector systems which are commercially availableand have ready applications herein include, but are not limited tomammalian cell systems infected with virus (e.g., vaccinia virus,adenovirus, etc.); insect cell systems infected with virus (e.g.,baculovirus); microorganisms such as yeast containing yeast vectors; orbacteria transformed with bacteriophage, DNA, plasmid DNA, or cosmidDNA. The expression elements of vectors vary in their strengths andspecificities. Depending on the host-vector system utilized, any one ofa number of suitable transcription and translation elements may be used.

[0456] An isolated nucleic acid molecule of the invention may beexpressed chromosomally, after integration of the coding sequence byrecombination. In this regard, any of a number of amplification systemsmay be used to achieve high levels of stable gene expression (SeeSambrook et al., 1989, supra).

[0457] A unicellular host transformed or transfected with an expressionvector of the invention can be cultured in an appropriate cell culturemedium that provides for expression of an isolated nucleic acid moleculeof the invention inserted into an expression vector and operativelyassociated with a promoter.

[0458] Any of the methods previously described for the insertion of DNAfragments into a cloning vector may be used to construct expressionvectors of the present invention. These methods may include in vitrorecombinant DNA and synthetic techniques and in vivo recombination(genetic recombination).

[0459] Expression of an isolated nucleic acid molecule of the inventionto produce a protein of the invention may be controlled by anypromoter/enhancer element known in the art. However, these regulatoryelements must be functional in the host selected for expression.Promoters which may be used to control expression include, but are notlimited to, the SV40 early promoter region (Benoist and Chambon, 1981,Nature 290:304-310), the promoter contained in the 3′ long terminalrepeat of Rous sarcoma virus (Yamamoto, et al., 1980, Cell 22:787-797),the herpes thymidine kinase promoter (Wagner et al., 1981, Proc. Natl.Acad. Sci. U.S.A. 78:1441-1445), the regulatory sequences of themetallothionein gene (Brinster et al., 1982, Nature 296:39-42);prokaryotic expression vectors such as the β-lactamase promoter(Villa-Kamaroff, et al., 1978, Proc. Natl. Acad. Sci. U.S.A.75:3727-3731), or the tac promoter (DeBoer, et al., 1983, Proc. Natl.Acad. Sci. U.S.A. 80:21-25); see also “Useful proteins from recombinantbacteria” in Scientific American, 1980, 242:74-94; promoter elementsfrom yeast or other fungi such as the Gal 4 promoter, the ADC (alcoholdehydrogenase) promoter, PGK (phosphoglycerol kinase) promoter, alkalinephosphatase promoter; and the animal transcriptional control regions,which exhibit tissue specificity and have been utilized in transgenicanimals: elastase I gene control region which is active in pancreaticacinar cells (Swift et al., 1984, Cell 38:639-646; Ornitz et al., 1986,Cold Spring Harbor Symp. Quant. Biol. 50:399-409; MacDonald, 1987,Hepatology 7:425-515); insulin gene control region which is active inpancreatic beta cells (Hanahan, 1985, Nature 315:115-122),immunoglobulin gene control region which is active in lymphoid cells(Grosschedl et al., 1984, Cell 38:647-658; Adames et al., 1985, Nature318:533-538; Alexander et al., 1987, Mol. Cell. Biol. 7:1436-1444),mouse mammary tumor virus control region which is active in testicular,breast, lymphoid and mast cells (Leder et al., 1986, Cell 45:485-495),albumin gene control region which is active in liver (Pinkert et al.,1987, Genes and Devel. 1:268-276), alpha-fetoprotein gene control regionwhich is active in liver (Krumlauf et al., 1985, Mol. Cell. Biol.5:1639-1648; Hammer et al., 1987, Science 235:53-58), alpha1-antitrypsin gene control region which is active in the liver (Kelseyet al., 1987, Genes and Devel. 1:161-171), beta-globin gene controlregion which is active in myeloid cells (Mogram et al., 1985, Nature315:338-340; Kollias et al., 1986, Cell 46:89-94), myelin basic proteingene control region which is active in oligodendrocyte cells in thebrain (Readhead et al., 1987, Cell 48:703-712), myosin light chain-2gene control region which is active in skeletal muscle (Sani, 1985,Nature 314:283-286), and gonadotropic releasing hormone gene controlregion which is active in the hypothalamus (Mason et al., 1986, Science234:1372-1378).

[0460] Moreover, an expression vector can be identified by four generalapproaches: (a) PCR amplification of the desired plasmid DNA or specificmRNA, (b) nucleic acid hybridization, (c) presence or absence ofselection marker gene functions, and (d) expression of insertedsequences. In the first approach, an isolated nucleic acid molecule ofthe invention can be amplified by PCR to provide for detection of theamplified product. In the second approach, the presence of a foreigngene inserted in an expression vector of the present invention can bedetected by nucleic acid hybridization using probes comprising sequencesthat are homologous to an inserted marker gene. In the third approach,the recombinant vector/host system can be identified and selected basedupon the presence or absence of certain “selection marker” genefunctions (e.g., β-galactosidase activity, thymidine kinase activity,resistance to antibiotics, transformation phenotype, occlusion bodyformation in baculovirus, etc.) caused by the insertion of foreign genesin the vector. In yet another example, if, an isolated nucleic acidmolecule of the invention is inserted within the “selection marker” genesequence of the vector, recombinants containing the insert can beidentified by the absence of the inserted gene function. In the fourthapproach, recombinant expression vectors can be identified by assayingfor the activity, biochemical, or immunological characteristics of thegene product expressed by the recombinant expression vector, providedthat the expressed protein assumes a functionally active conformation.

[0461] Naturally, the present invention extends to a method of producingan isolated protein of the invention. An example of such a methodcomprises the steps of culturing a unicellular host transformed ortransfected with an expression vector of the invention under conditionsthat provide for expression of the isolated nucleic acid molecule of theinvention inserted into the expression vector, to produce a protein ofthe invention. The protein produced depends upon which isolated nucleicacid molecule of the invention was inserted into the expression vector.The isolated protein produced can then be readily recovered from theunicellular host, the culture, or both.

[0462] A wide variety of unicellular host/expression vector combinationscommercially available to the skilled artisan may be employed inproducing an isolated protein of the invention.

[0463] Useful expression vectors, for example, may consist of segmentsof chromosomal, non-chromosomal and synthetic DNA sequences. Suitablevectors include derivatives of SV40 and known bacterial plasmids, e.g.,E. coli plasmids col E1, pCR1, pBR322, pMa1-C2, pET, pGEX (Smith et-al,1988, Gene 67:31-40), pMB9 and their derivatives, plasmids such as RP4;phage DNAS, e.g., the numerous derivatives of phage λ, e.g., NM989, andother phage DNA, e.g., M13 and filamentous single stranded phage DNA;yeast plasmids such as the 2μ plasmid or derivatives thereof; vectorsuseful in eukaryotic cells, such as vectors useful in insect ormammalian cells; vectors derived from combinations of plasmids and phageDNAs, such as plasmids that have been modified to employ phage DNA orother expression control sequences; and the like.

[0464] For example, in baculovirus expression systems, both non-fusiontransfer vectors, such as but not limited to pVL941 (BamH1 cloning site;Summers), pVL1393 (BamH1, SmaI, XbaI, EcoR1, NotI, XmaIII, BglII, andPstI cloning site; Invitrogen), pVL1392 (BglII, PstI, NotI, XmaIII,EcoR1, XbaI, SmaI, and BamH1 cloning site; Summers and Invitrogen), andpBlueBacIII (BamH1, BglII, PstI, NcoI, and HindIII cloning site, withblue/white recombinant screening possible; Invitrogen), and fusiontransfer vectors, such as but not limited to pAc700 (BamH1 and KpnIcloning site, in which the BamH1 recognition site begins with theinitiation codon; Summers), pAc70l and pAc702 (same as pAc700, withdifferent reading frames), pAc360 (BamH1 cloning site 36 base pairsdownstream of a polyhedrin initiation codon; Invitrogen(195)), andpBlueBacHisA, B, C (three different reading frames, with BamH1, BglII,PstI, NcoI, and HindIII cloning site, an N-terminal peptide for ProBondpurification, and blue/white recombinant screening of plaques;Invitrogen (220) can be used.

[0465] Mammalian expression vectors contemplated for use in theinvention include vectors with inducible promoters, such as thedihydrofolate reductase (DHFR) promoter, e.g., any expression vectorwith a DHFR expression vector, or a DHFR/methotrexate co-amplificationvector, such as pED PstI, SalI, SbaI, SmaI, and EcoRI cloning site, withthe vector expressing both the cloned gene and DHFR; see Kaufman,Current Protocols in Molecular Biology, 16.12(1991).

[0466] Alternatively, a glutamine synthetase/methionine sulfoximineco-amplification vector, such as pEE14 (HindIII, XbaI, SmaI, SbaI,EcoRI, and BclI cloning site, in which the vector expresses glutaminesynthase and the cloned gene; Celltech). In another embodiment, a vectorthat directs episomal expression under control of Epstein Barr Virus(EBV) can be used, such as pREP4 (BamH1, SfiI, XhoI, NotI, NheI,HindIII, AheI, PvuII, and KpnI cloning site, constitutive RSV-LTRpromoter, hygromycin selectable marker; Invitrogen), pCEP4 (BamH1, SfiI,XhoI, NotI, NheI, HindIII, NheI, PvuII, and KpnI cloning site,constitutive hCMV immediate early gene, hygromycin selectable marker;Invitrogen), pMEP4 (KpnI, PvuI, NheI, HindIII, NotI, XhoI, SfiI, BamH1cloning site, inducible metallothionein IIa gene promoter, hygromycinselectable marker: Invitrogen), pREP8 (BamH1, XhoI, NotI, HindIII, NheI,and KpnI cloning site, RSV-LTR promoter, histidinol selectable marker;Invitrogen), pREP9 (KpnI, NheI, HindIII, NotI, XhoI, SfiI, and BamHIcloning site, RSV-LTR promoter, G418 selectable marker; Invitrogen), andpEBVHis (RSV-LTR promoter, hygromycin selectable marker, N-terminalpeptide purifiable via ProBond resin and cleaved by enterokinase;Invitrogen). Selectable mammalian expression vectors for use in theinvention include pRc/CMV (HindIII, BstXI, NotI, SbaI, and ApaI cloningsite, G418 selection; Invitrogen), pRc/RSV (HindIII, SpeI, BstXI, NotI,XbaI cloning site, G418 selection; Invitrogen), and others. Vacciniavirus mammalian expression vectors (see, Kaufman, 1991, supra) for useaccording to the invention include but are not limited to pSC11 (SmaIcloning site, TK- and β-gal selection), pMJ601 (SalI, SmaI, AflI, NarI,BspMII, BamHI, ApaI, NheI, SaclI, KpnI, and HindIII cloning site; TK-and β-gal selection), and pTKgptF1S (EcoR1, PstI, SalI, AccI, HindIII,SbaI, BamHI, and Hpa cloning site, TK or XPRT selection).

[0467] Yeast expression systems can also be used according to theinvention include the non-fusion pYES2 vector (XbaI, SphI, ShoI, NotI,GstXI, EcoRI, BstXI, BamHI, Saci, KpnI, and HindIII cloning sit;Invitrogen) or the fusion pYESHisA, B, C (XbaI, SphI, ShoI, NotI, BstXI,EcoR1, BamH 1, SacI, KpnI, and HindIII cloning site, N-terminal peptidepurified with ProBond resin and cleaved with enterokinase; Invitrogen),to mention just two, can be employed according to the invention.

[0468] Once a particular recombinant DNA molecule is identified andisolated, several methods known in the art may be used to propagate it.Furthermore, once a suitable host system and growth conditions areestablished, recombinant expression vectors can be propagated andprepared in quantity. As previously explained, the expression vectorswhich can be used include, but are not limited to, the following vectorsor their derivatives: human or animal viruses such as vaccinia virus oradenovirus; insect viruses such as baculovirus; yeast vectors;bacteriophage vectors (e.g., lambda), and plasmid and cosmid DNAvectors, to name but a few.

[0469] Examples of unicellular hosts contemplated by the presentinvention, which are well know to those of ordinary skill in the art,include but are not limited to, E. coli Pseudonomas, Bacillus,Strepomyces, yeast, CHO, R1.1, B-W, L-M, COS1, COS7, BSC1, BSC40, BMT10,or HeLa cells, all of which are readily available to the skilledartisan. In addition, a host cell strain may be chosen which modulatesthe expression of an isolated nucleic acid molecule of the inventionmodifies and processes the gene product in the specific fashion desired.Different host cells have characteristic and specific mechanisms for thetranslational and post-translational processing and modification (e.g.,glycosylation, cleavage [e.g., of signal sequence]) of proteins.Appropriate cell lines or host systems can be chosen to ensure thedesired modification and processing of the foreign protein expressed.For example, expression in a bacterial system can be used to produce annonglycosylated core protein product. However, a translocation signalsequence of the product produced from expression of an isolated nucleicacid molecule of the invention in bacteria may not be properly spliced.Expression in yeast can produce a glycosylated product. Expression ineukaryotic cells can increase the likelihood of “native” glycosylationand folding. Moreover, expression in mammalian cells can provide a toolfor reconstituting, or constituting activity of murine prolinedehydrogenase, human PRODH, or a variant human PRODH protein of theinvention. Furthermore, different vector/host expression systems mayaffect processing reactions, such as proteolytic cleavages, to adifferent extent.

[0470] Vectors are introduced into the desired unicellular hosts bymethods known in the art, e.g., transfection, electroporation,microinjection, transduction, cell fusion, DEAE dextran, calciumphosphate precipitation, lipofection (lysosome fusion), use of a genegun, or a DNA vector transporter (see, e.g., Wu et al., 1992, J. Biol.Chem. 267:963-967; Wu and Wu, 1988, J. Biol. Chem. 263:14621-14624;Hartmut et al., Canadian Patent Application No. 2,012,311, filed Mar.15, 1990).

Antibodies to an Isolated Protein of the Invention

[0471] According to the invention, an isolated protein of the inventionmay be used as an immunogen to generate antibodies. Such antibodiesinclude but are not limited to polyclonal, monoclonal, chimeric, singlechain, Fab fragments, and an Fab expression library. Furthermore,antibodies of the invention may be cross reactive, e.g., they mayrecognize more than one isolated protein of the invention.

[0472] Various procedures known in the art may be used for theproduction of polyclonal antibodies of the invention. For the productionof antibody, various host animals can be immunized by injection with animmunogen described above. Examples of such animals include, but are notlimited to rabbits, mice, rats, sheep, goats, etc. In one embodiment, anisolated protein of the invention can be conjugated to an immunogeniccarrier, e.g., bovine serum albumin (BSA) or keyhole limpet hemocyanin(KLH). Various adjuvants may be used to increase the immunologicalresponse, depending on the host species, including but not limited toFreund's (complete and incomplete), mineral gels such as aluminumhydroxide, surface active substances such as lysolecithin, pluronicpolyols, polyanions, peptides, oil emulsions, keyhole limpethemocyanins, dinitrophenol, and potentially useful human adjuvants suchas BCG (bacille Calmette-Guerin) and Corynebacterium parvum.

[0473] For preparation of monoclonal antibodies of the invention, anytechnique that provides for the production of antibody molecules bycontinuous cell lines in culture may be used. These include but are notlimited to the hybridoma technique originally developed by Kohler andMilstein [Nature 256:495-497 (1975)], as well as the trioma technique,the human B-cell hybridoma technique [Kozbor et al., Immunology Today4:72 1983); Cote et al., Proc. Natl. Acad. Sci. U.S.A. 80:2026-2030(1983)], and the EBV-hybridoma technique to produce human monoclonalantibodies [Cole et al., in Monoclonal Antibodies and Cancer Therapy,Alan R. Liss, Inc., pp. 77-96 (1985)]. In an additional embodiment ofthe invention, monoclonal antibodies can be produced in germ-freeanimals utilizing technology set forth in PCT/US90/02545. In fact,according to the invention, techniques developed for the production of“chimeric antibodies” [Morrison et al., J. Bacteriol. 159:870 (1984);Neuberger et al., Nature 312:604-608 (1984); Takeda et al., Nature314:452-454 (1985)] by splicing the genes from a mouse which encode anantibody molecule specific for a protein of the invention together withgenes from a human antibody molecule of appropriate biological activitycan be used, and are within the scope of this invention. Such human orhumanized chimeric antibodies are preferred for use in determining thepresence of an isolated protein of the invention in a sample.

[0474] According to the invention, techniques described for theproduction of single chain antibodies [U.S. Pat. Nos. 5,476,786 and5,132,405 to Huston; U.S. Pat. No. 4,946,778] can be adapted to produceparticular isolated variant human PRODH protein-specific single chainantibodies. An additional embodiment of the invention utilizes thetechniques described for the construction of Fab expression libraries[Huse et al., Science 246:1275-1281 (1989)] to allow rapid and easyidentification of monoclonal Fab fragments with the desired specificityfor an isolated protein of the invention.

[0475] Antibody fragments which contain the idiotype of the antibodymolecule can be generated by known techniques. For example, suchfragments include but are not limited to: the F(ab′)₂ fragment which canbe produced by pepsin digestion of the antibody molecule; the Fab′fragments which can be generated by reducing the disulfide bridges ofthe F(ab′)₂ fragment, and the Fab fragments which can be generated bytreating the antibody molecule with papain and a reducing agent.

[0476] In the production of antibodies, screening for the desiredantibody can be accomplished by techniques known in the art, e.g.,radioimmunoassay, ELISA (enzyme-linked immunosorbant assay), “sandwich”immunoassays, immunoradiometric assays, gel diffusion precipitinreactions, immunodiffusion assays, in situ immunoassays (using colloidalgold, enzyme or radioisotope labels, for example), western blots,precipitation reactions, agglutination assays (e.g., gel agglutinationassays, hemagglutination assays), complement fixation assays,immunofluorescence assays, protein A assays, and immunoelectrophoresisassays, etc. In one embodiment, antibody binding is detected bydetecting a label on the primary antibody. In another embodiment, theprimary antibody is detected by detecting binding of a secondaryantibody or reagent to the primary antibody. In a further embodiment,the secondary antibody is labeled. Many means are known in the art fordetecting binding in an immunoassay and are within the scope of thepresent invention. For example, to select antibodies which recognize aspecific epitope of an isolated protein of the invention, one may assaygenerated hybridomas for a product which binds to a fragment of avariant PRODH protein containing such epitope.

[0477] The foregoing antibodies can be used in methods known in the artrelating to the localization and activity of an isolated variant humanPRODH protein, conservative variants thereof, or fragments thereof e.g.,for Western blotting, imaging a variant human PRODH protein in situ,measuring levels thereof in appropriate physiological samples, etc.using any of the detection techniques mentioned above or known in theart.

Commercial Kits

[0478] In a further embodiment, the present invention extends tocommercial test kits suitable for use by a medical professional todetermine the presence or absence of predetermined PRODH activity, orpredetermined PRODH capability in target patient populations.

[0479] In accordance with the testing techniques discussed above, oneclass of such kits comprise at least the labeled PRODH or its bindingpartner, for instance an antibody specific thereto, and directions, ofcourse, depending upon the method selected, e.g., “competitive,”“sandwich,” “DASP” and the like. The kits may also contain peripheralreagents such as buffers, stabilizers, etc. In a particular embodiment,the labeled PRODH comprises an amino acid sequence of SEQ ID NO:2.

[0480] Another class of such kits may also include PCR reagents, such asoligonucleotide primers, enzymes, gel matrixes, buffers, etc.

[0481] Accordingly, a test kit may be prepared for the diagnosis ordetection of a susceptibility to schizophrenia, or a disease or disorderrelated thereto, to measure levels of PRODH activity in a bodily samplefrom a subject, wherein the kit comprises:

[0482] (a) a predetermined amount of at least one labeledimmunochemically reactive component obtained by the direct or indirectattachment of the present PRODH factor or a specific binding partnerthereto, to a detectable label;

[0483] (b) other reagents; and

[0484] (c) directions, including comparison levels of PRODH, for use ofthe kit.

[0485] An alternate kit for measuring the levels of PRODH activity maycomprise PCR reagents, such as oligonucleotide primers, enzymes, gelmatrices, buffers, directions, including comparison levels of PRODH, foruse of the kit. A still further alternate can utilize reagents formeasuring the levels of PRODH activity and directions, includingcomparison levels of PRODH for use of the kit.

[0486] In a further variation, a test kit may be prepared and used forthe purposes stated above, which operates according to a predeterminedprotocol (e.g. “competitive,” “sandwich,” “double antibody,” etc.), andcomprises:

[0487] (a) a labeled component which has been obtained by coupling PRODHcomprising an amino acid sequence of SEQ ID NO:2, a conservative variantthereof, or fragment thereof to a detectable label;

[0488] (b) one or more additional immunochemical reagents of which atleast one reagent is a ligand or an immobilized ligand, which ligand isselected from the group consisting of:

[0489] (i) a ligand capable of binding with the labeled component (a);

[0490] (ii) a ligand capable of binding with a binding partner of thelabeled component (a);

[0491] (iii) a ligand capable of binding with at least one of thecomponent(s) to be determined; and

[0492] (iv) a ligand capable of binding with at least one of the bindingpartners of at least one of the component(s) to be determined; and

[0493] (c) directions for the performance of a protocol for thedetection and/or determination of one or more components of animmunochemical reaction between PRODH and a specific binding partnerthereto.

[0494] Other kits of the present invention utilize the discovery that,unexpectedly, the presence of a particular variant allele of the PRODHgene in the genome of a subject is indicative of an increasedsusceptibility to schizophrenia or a disease or disorder relatedthereto, or a diagnosis of schizophrenia in the subject. Hence, thepresent invention extends to a test kit to facilitate diagnosis andtreatment of schizophrenia or a disease or disorder related thereto in aeukaryotic cellular sample, wherein the test kit comprises:

[0495] a) PCR oligonucleotide primers suitable for detection of anisolated variant allele of a PRODH gene, wherein the PRODH genecomprises a DNA sequence of SEQ ID NO:1, and the variant allelecomprises a DNA sequence having a variation in SEQ ID NO:1 comprising atransition of A to G in the second position of codon 437 of SEQ ID NO:1;

[0496] (b) other reagents such as enzymes, gel matrices, buffers, etc.;and

[0497] (c) directions for use of the kit.

[0498] Assays for Screening the Ability Drugs and Therapeutic Agents toTreat Schizophrenia or a Disease or Disorder Related Thereto

[0499] In accordance with the above, an assay system for screeningpotential drugs effective to modulate levels of human PRODH in a subjectmay be prepared. The PRODH may be introduced into a test system, such asa cell culture, and the prospective drug may also be introduced into thecell culture. The cell culture is then examined to observe any changesin the PRODH activity of the cells, due either to the addition of theprospective drug alone, or due to the effect of added quantities of theknown PRODH.

[0500] Moreover, the present invention extends to an assay system forscreening drugs and other agents for their ability to treatschizophrenia or a disease or disorder related thereto. Such an assay ofthe present invention comprises the steps of culturing an observablecellular test colony inoculated with the drug or agent to be assayed,harvesting a cellular extract from the cellular test colony, andexamining the extract for the presence of PRODH. An increase or decreasein the level of activity of PRODH in the test colony compared to thelevel of activity of PRODH in a control colony indicates the ability ofthe drug to modulate the production, stability, degradation or activityof PRODH, which is indicative of the ability of the drug or agent totreat schizophrenia or a disease or disorder related thereto, such asobsessive compulsive disorder (OCD), bipolar disorder (BP) or majordepressive disorder. An increase in the level of activity of PRODH inthe test colony compared to level of activity in a control colonyindicates the drug or agent is a potential therapeutic agent for thetreatment of schizophrenia or a disease or disorder related thereto.

[0501] In another embodiment, the present invention extends to an assaysystem for screening drugs, agents or compounds, to determine theirschizophrenic-related pharmacological activity. An example of such amethod comprises the steps of:

[0502] administering the compound to a mammal;

[0503] determining the level of activity of PRODH in the mammal; and

[0504] comparing the level of activity of PRODH to the level of activityof PRODH in a control animal to which the compound was not administered.An increase in the level of activity of PRODH in the mammal relative tothe level of PRODH activity in the standard is indicative that the drug,agent or compound may have schizophrenic-related pharmacologicalactivity, and ability as a therapeutic agent for treating schizophreniaor a disease or disorder related thereto, such as obsessive compulsivedisorder (OCD), bipolar disorder (BP) or major depressive disorder.

[0505] Another example of an assay system for screening drugs, agents orcompounds for schizophrenic related pharmacological activity comprisesthe steps of:

[0506] determining a basal level of activity of PRODH in a mammal;

[0507] administering the compound to the mammal;

[0508] determining the level of activity of PRODH in the mammal afteradministration of the compound. An increase in the level of activity ofPRODH in the mammal relative to the basal level of activity indicatesthe compound has a schizophrenic-related pharmacological activity, andmay have potential as a therapeutic agent for treating schizophrenia ora disease or disorder related thereto, such as obsessive compulsivedisorder (OCD), bipolar disorder (BP), or major depressive disorder(MDD).

[0509] Methods of assaying levels of activity of PRODH are readily knownto one of ordinary skill in the art, and are disclosed in Blake, R. &Russel, E., Hyperprolinemia and prolinuria in a new inbred strain ofmice PRO/Re. Science 176, 809-811 (1972); Blake, R. L., Animal Model forHyperprolinaemia: Deficiency of Mouse Proline Oxidase Activity Biochem.J. 129, 987-989 (1979) both of which are hereby incorporated byreference in their entireties.

Treating Schizophrenia or a Disease or Disorder Related Thereto

[0510] In another embodiment, the present invention extends to a methodof treating schizophrenia or a disease or disorder related thereto, suchas obsessive compulsive disorder (OCD), bipolar disorder (BP), or majordepressive disorder (MDD) in a subject, or a symptom of such a diseaseor disorder. An example of such a method comprises administering to thesubject a therapeutically effective amount of isolated PRODH comprisingan amino acid sequence of SEQ ID NO:2, a conservative variant thereof,fragment thereof, or analog or derivative thereof.

[0511] Generally, the PRODH protein of the present invention may bederivatized by the attachment of one or more chemical moieties to theprotein moiety. The chemically modified derivatives may be furtherformulated for intraarterial, intraperitoneal, intramuscularsubcutaneous, intravenous, oral, nasal, pulmonary, topical or otherroutes of administration. Chemical modification of biologically activecomponent or components may provide additional advantages under certaincircumstances, such as increasing the stability and circulation time ofthe component or components and decreasing immunogenicity. See U.S. Pat.No. 4,179,337, Davis et al., issued Dec. 18, 1979. For a review, seeAbuchowski et al., in Enzymes as Drugs (J. S. Holcerberg and J. Roberts,eds. pp. 367-383 (1981)). A review article describing proteinmodification and fusion proteins is Francis, 1992, Focus on GrowthFactors 3:4-10, Mediscript: Mountview Court, Friem Barnet Lane, LondonN20, OLD, UK.

[0512] Chemical Moieties For Derivatization.

[0513] The chemical moieties suitable for denvatization may be selectedfrom among water soluble polymers. The polymer selected should be watersoluble so that the component to which it is attached does notprecipitate in an aqueous environment, such as a physiologicalenvironment. Preferably, for therapeutic use of the end-productpreparation, the polymer will be pharmaceutically acceptable. Oneskilled in the art will be able to select the desired polymer based onsuch considerations as whether the polymer/component conjugate will beused therapeutically, and if so, the desired dosage, circulation time,resistance to proteolysis, and other considerations. For the presentcomponent or components, these may be ascertained using the assaysprovided herein.

[0514] The water soluble polymer may be selected from the groupconsisting of, for example, polyethylene glycol, copolymers of ethyleneglycol/propylene glycol, carboxymethylcellulose, dextran, polyvinylalcohol, polyvinyl pyrrolidone, poly-¹, 3-dioxolane,poly-1,3,6-trioxane, ethylene/maleic anhydride copolymer, polyaminoacids(either homopolymers or random copolymers), and dextran or poly(n-vinylpyrrolidone)polyetlylene glycol, propropylene glycol homopolymers,prolypropylene oxide/ethylene oxide co-polymers, polyoxyethylatedpolyols and polyvinyl alcohol. Polyethylene glycol propionaldenhyde mayhave advantages in manufacturing due to its stability in water.

[0515] The polymer may be of any molecular weight, and may be branchedor unbranched. For polyethylene glycol, the preferred molecular weightis between about 2 kDa and about 100 kDa (the term “about” indicatingthat in preparations of polyethylene glycol, some molecules will weighmore, some less, than the stated molecular weight) for ease in handlingand manufacturing. Other sizes may be used, depending on the desiredtherapeutic profile (e.g., the duration of sustained release desired,the effects, if any on biological activity, the ease in handling, thedegree or lack of antigenicity and other known effects of thepolyethylene glycol to a therapeutic protein or analog).

[0516] The number of polymer molecules so attached may vary, and oneskilled in the art will be able to ascertain the effect on function. Onemay mono-derivatize, or may provide for a di-, tri-, tetra- or somecombination of derivatization, with the same or different chemicalmoieties (e.g., polymers, such as different weights of polyethyleneglycols). The proportion of polymer molecules to component or componentsmolecules will vary, as will their concentrations in the reactionmixture. In general, the optimum ratio (in terms of efficiency ofreaction in that there is no excess unreacted component or componentsand polymer) will be determined by factors such as the desired degree ofderivatization (e.g., mono, di-, tri-, etc.), the molecular weight ofthe polymer selected, whether the polymer is branched or unbranched, andthe reaction conditions.

[0517] The polyethylene glycol molecules (or other chemical moieties)should be attached to the component or components with consideration ofeffects on functional or antigenic domains of the protein. There are anumber of attachment methods available to those skilled in the art,e.g., EP 0 401 384 herein incorporated by reference (coupling PEG toG-CSF), see also Malik et al., 1992, Exp. Hematol. 20:1028-1035(reporting pegylation of GM-CSF using tresyl chloride). For example,polyethylene glycol may be covalently bound through amino acid residuesvia a reactive group, such as, a free amino or carboxyl group. Reactivegroups are those to which an activated polyethylene glycol molecule maybe bound. The amino acid residues having a free amino group includelysine residues and the—terminal amino acid residues; those having afree carboxyl group include aspartic acid residues glutamic acidresidues and the C-terminal amino acid residue. Sulfhydryl groups mayalso be used as a reactive group for attaching the polyethylene glycolmolecule(s). Preferred for therapeutic purposes is attachment at anamino group, such as attachment at the N-terminus or lysine group.

[0518] One may specifically desire N-terminally chemically modifiedprotein. Using polyethylene glycol as an illustration of the presentcompositions, one may select from a variety of polyethylene glycolmolecules (by molecular weight, branching, etc.), the proportion ofpolyethylene glycol molecules to protein (or peptide) molecules in thereaction mix, the type of pegylation reaction to be performed, and themethod of obtaining the selected N-terminally pegylated protein. Themethod of obtaining the N-terminally pegylated preparation (i.e.,separating this moiety from other monopegylated moieties if necessary)may be by purification of the N-terminally pegylated material from apopulation of pegylated protein molecules. Selective N-terminalchemically modification may be accomplished by reductive alkylationwhich exploits differential reactivity of different types of primaryamino groups (lysine versus the N-terminal) available for derivatizationin a particular protein. Under the appropriate reaction conditions,substantially selective derivatization of the protein at the N-terminuswith a carbonyl group containing polymer is achieved. For example, onemay selectively N-terminally pegylate the protein by performing thereaction at pH which allows one to take advantage of the pK₁ differencesbetween the e-amino groups of the lysine residues and that of thea-amino group of the N-terminal residue of the protein. By suchselective derivatization attachment of a water soluble polymer to aprotein is controlled: the conjugation with the polymer takes placepredominantly at the N-terminus of the protein and no significantmodification of other reactive groups, such as the lysine side chainamino groups, occurs. Using reductive alkylation, the water solublepolymer may be of the type described above, and should have a singlereactive aldehyde for coupling to the protein. Polyethylene glycolproprionaldehyde containing a single reactive aldehyde, may be used.

Pharmaceutical Compositions

[0519] In yet another aspect of the present invention, provided arepharmaceutical compositions comprising a protein comprising an aminoacid sequence of SEQ ID NO:2, conservative variants thereof, fragmentsthereof, or analogs or derivatives thereof, and a pharmaceuticallyacceptable carrier thereof. Such pharmaceutical compositions may be foradministration for injection, or for oral, pulmonary, nasal or otherforms of administration. In general, comprehended by the invention arepharmaceutical compositions comprising effective amounts of a lowmolecular weight component or components, or derivative products, of theinvention together with pharmaceutically acceptable diluents,preservatives, solubilizers, emulsifiers, adjuvants and/or carriers.Such compositions include diluents of various buffer content (e.g.,Tris-HCl, acetate, phosphate), pH and ionic strength; additives such asdetergents and solubilizing agents (e.g., Tween 80, Polysorbate 80),anti-oxidants (e.g., ascorbic acid, sodium metabisulfite), preservatives(e.g., Thimersol, benzyl alcohol) and bulking substances (e.g., lactose,mannitol); incorporation of the material into particulate preparationsof polymeric compounds such as polylactic acid, polyglycolic acid. etc.or into liposomes. Hylauronic acid may also be used. Such compositionsmay influence the physical state, stability, rate of in vivo release,and rate of in vivo clearance of the present proteins and derivatives.See, e.g., Remington's Pharmaceutical Sciences, 18th Ed. (1990, MackPublishing Co., Easton, Pa. 18042) pages 1435-1712 which are hereinincorporated by reference. The compositions may be prepared in liquidform, or may be in dried powder, such as lyophilized form.

Oral Delivery

[0520] Contemplated for use herein are oral solid dosage forms, whichare described generally in Remington's Pharmaceutical Sciences, 18th Ed.1990 (Mack Publishing Co. Easton Pa. 18042) at Chapter 89, Which isherein incorporated by reference. Solid dosage forms include tablets,capsules, pills, troches or lozenges, cachets or pellets. Also,liposomal or proteinoid encapsulation may be used to formulate thepresent compositions (as, for example, proteinoid microspheres reportedin U.S. Pat. No. 4,925,673). Liposomal encapsulation may be used and theliposomes may be derivatized with various polymers (e.g., U.S. Pat. No.5,013,556). A description of possible solid dosage forms for thetherapeutic is given by Marshall, K. In: Modern Pharmaceutics Edited byG. S. Banker and C. T. Rhodes Chapter 10, 1979, herein incorporated byreference. In general, the formulation will include the component orcomponents (or chemically modified forms thereof) and inert ingredientswhich allow for protection against the stomach environment, and releaseof the biologically active material in the intestine.

[0521] Also specifically contemplated are oral dosage forms of the abovederivatized PRODH, conservative variant thereof, or fragment thereof.The PRODH, conservative variant thereof, or fragment thereof may bechemically modified so that oral delivery of the derivative isefficacious. Generally, the chemical modification contemplated is theattachment of at least one moiety to the component molecule itself,where said moiety permits (a) inhibition of proteolysis; and (b) uptakeinto the blood stream from the stomach or intestine. Also desired is theincrease in overall stability of the component or components andincrease in circulation time in the body. Examples of such moietiesinclude: polyethylene glycol, copolymers of ethylene glycol andpropylene glycol, carboxymethyl cellulose, dextran, polyvinyl alcohol,polyvinyl pyrrolidone and polyproline. Abuchowski and Davis, 1981,“Soluble Polymer-Enzyme Adducts” In: Enzymes as Drugs, Hocenberg andRoberts, eds., Wiley-Interscience, New York, N.Y., pp. 367-383; Newmark,et al., 1982, J. Appl. Biochem. 4:185-189. Other polymers that could beused are poly-13-dioxolane and poly-1,3,6-tioxocane. Preferred forpharmaceutical usage, as indicated above, are polyethylene glycolmoieties.

[0522] For the component (or derivative) the location of release may bethe stomach, the small intestine (the duodenum, the jejunum, or theileum), or the large intestine. One skilled in the art has availableformulations which will not dissolve in the stomach, yet will releasethe material in the duodenum or elsewhere in the intestine. Preferably,the release will avoid the deleterious effects of the stomachenvironment, either by protection of the protein (or derivative) or byrelease of the biologically active material beyond the stomachenvironment, such as in the intestine.

[0523] To ensure fill gastric resistance a coating impermeable to atleast pH 5.0 is essential. Examples of the more common inert ingredientsthat are used as enteric coatings are cellulose acetate trimellitate(CAT), hydroxypropylmethylcellulose phthalate (HPMCP), HPMCP 50, HPMCP55, polyvinyl acetate phthalate (PVAP), Eudragit L30D, Aquateric,cellulose acetate phthalate (CAP), Eudragit L, Eudragit S, and Shellac.These coatings may be used as mixed films.

[0524] A coating or mixture of coatings can also be used on tablets,which are not intended for protection against the stomach. This caninclude sugar coatings, or coatings which make the tablet easier toswallow. Capsules may consist of a hard shell (such as gelatin) fordelivery of dry therapeutic i.e. powder; for liquid forms, a softgelatin shell may be used. The shell material of cachets could be thickstarch or other edible paper. For pills, lozenges, molded tablets ortablet triturates, moist massing techniques can be used.

[0525] The therapeutic can be included in the formulation as finemulti-particulates in the form of granules or pellets of particle sizeabout 1 mm. The formulation of the material for capsule administrationcould also be as a powder, lightly compressed plugs or even as tablets.The therapeutic could be prepared by compression.

[0526] Colorants and flavoring agents may all be included. For example,the protein (or derivative) may be formulated (such as by liposome ormicrosphere encapsulation) and then further contained within an edibleproduct, such as a refrigerated beverage containing colorants andflavoring agents.

[0527] One may dilute or increase the volume of the therapeutic with aninert material. These diluents could include carbohydrates, especiallymannitol, a-lactose, anhydrous lactose, cellulose, sucrose, modifieddextrans and starch. Certain inorganic salts may be also be used asfillers including calcium triphosphate, magnesium carbonate and sodiumchloride. Some commercially available diluents are Fast-Flo, Emdex.STA-Rx 1500, Emcompress and Avicell.

[0528] Disintegrants may be included in the formulation of thetherapeutic into a solid dosage form. Materials used as disintegratesinclude but are not limited to starch, including the commercialdisintegrant based on starch, Explotab. Sodium starch glycolate,Amberlite, sodium carboxymethylcellulose, ultramylopectin, sodiumalginate, gelatin, orange peel acid carboxymethyl cellulose, naturalsponge and bentonite may all be used. Another form of the disintegrantsare the insoluble cationic exchange resins. Powdered gums may be used asdisintegrants and as binders and these can include powdered gums such asagar, Karaya or tragacanth. Alginic acid and its sodium salt are alsouseful as disintegrants.

[0529] Binders may be used to hold the therapeutic agent together toform a hard tablet and include materials from natural products such asacacia, tragacanth, starch and gelatin. Others include methyl cellulose(MC), ethyl cellulose (EC) and carboxymethyl cellulose (CMC). Polyvinylpyrrolidone (PVP) and hydroxypropylmethyl cellulose (HPMC) could both beused in alcoholic solutions to granulate the therapeutic.

[0530] An anti-frictional agent may be included in the formulation ofthe therapeutic to prevent sticking during the formulation process.Lubricants may be used as a layer between the therapeutic and the diewall, and these can include but are not limited to; stearic acidincluding its magnesium and calcium salts, polytetrafluoroethylene(PTFE), liquid paraffin, vegetable oils and waxes. Soluble lubricantsmay also be used such as sodium lauryl sulfate, magnesium laurylsulfate, polyethylene glycol of various molecular weights, Carbowax 4000and 6000.

[0531] Glidants that might improve the flow properties of the drugduring formulation and to aid rearrangement during compression might beadded. The glidants may include starch, talc, pyrogenic silica andhydrated silicoaluminate.

[0532] To aid dissolution of the therapeutic into the aqueousenvironment a surfactant might be added as a wetting agent. Surfactantsmay include anionic detergents such as sodium lauryl sulfate, dioctylsodium sulfosuccinate and dioctyl sodium sulfonate. Cationic detergentsmight be used and could include benzalkonium chloride or benzethomiumchloride. The list of potential non-ionic detergents that could beincluded in the formulation as surfactants are lauromacrogol 400,polyoxyl 40 stearate, polyoxyethylene hydrogenated castor oil 10, 50 and60, glycerol monostearate, polysorbate 40, 60, 65 and 80, sucrose fattyacid ester, methyl cellulose and carboxymethyl cellulose. Thesesurfactants could be present in the formulation of the protein orderivative either alone or as a mixture in different ratios.

[0533] Additives which potentially enhance uptake of the protein (orderivative) are for instance the fatty acids oleic acid, linoleic acidand linolenic acid.

[0534] Controlled release oral formulation may be desirable. The drugcould be incorporated into an inert matrix which permits release byeither diffusion or leaching mechanisms, e.g., gums.

[0535] Slowly degenerating matrices may also be incorporated into theformulation. Some enteric coatings also have a delayed release effect.Another form of a controlled release of this therapeutic is by a methodbased on the Oros therapeutic system (Alza Corp.), i.e. the drug isenclosed in a semipermeable membrane which allows water to enter andpush drug out through a single small opening due to osmotic effects.

[0536] Other coatings may be used for the formulation. These include avariety of sugars which could be applied in a coating pan. Thetherapeutic agent could also be given in a film coated tablet and thematerials used in this instance are divided into 2 groups. The first arethe nonenteric materials and include methyl cellulose, ethyl cellulose,Methydroxyethyl cellulose, methylhydroxyethyl cellulose, hydroxypropylcellulose, hydroxypropyl-methyl cellulose, sodium carboxymethylcellulose, providone and the polyethylene glycols. The second groupconsists of the enteric materials that are commonly esters of phthalicacid.

[0537] A mix of materials might be used to provide the optimum filmcoating. Film coating may be carried out in a pan-coater or in afluidized bed or by compression coating.

[0538] Pulmonary Delivery.

[0539] Also contemplated herein is pulmonary delivery of the presentprotein (or derivatives thereof). The protein (or derivative) isdelivered to the lungs of a mammal while inhaling and traverses acrossthe lung epithelial lining to the blood stream. Other reports of thisinclude Adjei et al., 1990, Pharmaceutical Research, 7:565-569; Adjei etal., 1990, International Journal of Pharmaceutics, 63:135-144(leuprolide acetate); Braquet et al., 1989, Journal of CardiovascularPharmacology, 13(suppl. 5):143-146 (endothelin-l). Hubbard et al., 1989,Annals of Internal Medicine, Vol. III, pp. 206-212 (a1-antitryspin);Smith et al., 1989, J. Clin. Invest. 84:1145-1146 (a-1-proteinase);Oswein et al., 1990, “Aerosolization of Proteins”, Proceedings ofSymposium on Respiratory Drug Delivery II, Keystone, Colo., March,(recombinant human growth hormone); Debs et al., 1988, J. Immunol.140:3482-3488 (interferon-g and tumor necrosis factor alpha) and Platzet al. U.S. Pat. No. 5,284,656 (granulocyte colony stimulating factor).A method and composition for pulmonary delivery of drugs for systemiceffect is described in U.S. Pat. No. 5,451,569, issued Sep. 19, 1995 toWong et al.

[0540] Contemplated for use in the practice of this invention are a widerange of mechanical devices designed for pulmonary delivery oftherapeutic products, including but not limited to nebulizers, metereddose inhalers, and powder inhalers, all of which are familiar to thoseskilled in the art.

[0541] Some specific examples of commercially available devices suitablefor the practice of this invention are the Ultravent nebulizer,manufactured by Mallinckrodt, Inc., St. Louis, Mo.; the Acorn IInebulizer, manufactured by Marquest Medical Products, Englewood, Colo.;the Ventolin metered dose inhaler, manufactured by Glaxo Inc., ResearchTriangle Park, North Carolina; and the Spinhaler powder inhaler,manufactured by Fisons Corp. Bedford, Mass.

[0542] All such devices require the use of formulations suitable for thedispensing of protein (or derivative). Typically, each formulation isspecific to the type of device employed and may involve the use of anappropriate propellant material, in addition to the usual diluents,adjuvants and/or carriers useful in therapy. Also, the use of liposomes,microcapsules or microspheres, inclusion complexes, or other types ofcarriers is contemplated. Chemically modified protein may also beprepared in different formulations depending on the type of chemicalmodification or the type of device employed.

[0543] Formulations suitable for use with a nebulizer, either jet orultrasonic, will typically comprise protein (or derivative) dissolved inwater at a concentration of about 0.1 to 25 mg of biologically activeprotein per mL of solution. The formulation may also include a bufferand a simple sugar (e.g., for protein stabilization and regulation ofosmotic pressure). The nebulizer formulation may also contain asurfactant, to reduce or prevent surface induced aggregation of theprotein caused by atomization of the solution in forming the aerosol.

[0544] Formulations for use with a metered-dose inhaler device willgenerally comprise a finely divided powder containing the protein (orderivative) suspended in a propellant with the aid of a surfactant. Thepropellant may be any conventional material employed for this purpose,such as a chlorofluorocarbon, a hydrochlorofluorocarbon, ahydrotluorocarbon, or a hydrocarbon, including trichlorofluoromethane,dichlorodifluoromethane, dichlorotetrafluoroethanol, and1,1,1,2-tetrafluoroethane, or combinations thereof. Suitable surfactantsinclude sorbitan trioleate and soya lecithin. Oleic acid may also beuseful as a surfactant.

[0545] Formulations for dispensing from a powder inhaler device willcomprise a finely divided dry powder containing protein (or derivative)and may also include a bulking agent, such as lactose, sorbitol,sucrose, or mannitol in amounts which facilitate dispersal of the powderfrom the device, e.g., 50 to 90% by weight of the formulation. Theprotein (or derivative) should most advantageously be prepared inparticulate form with an average particle size of less than 10 nm (ormicrons), most preferably 0.5 to 5 mm, for most effective delivery tothe distal lung.

[0546] Nasal Delivery.

[0547] Nasal delivery of the protein (or derivative) is alsocontemplated. Nasal delivery allows the passage of the protein to theblood stream directly after administering the therapeutic product to thenose, without the necessity for deposition of the product in the lung.

[0548] Formulations for nasal delivery include those with dextran orcyclodextran.

Methods of Treatment, Methods of Preparing a Medicament

[0549] In another embodiment, the present invention extends to a methodof treating schizophrenia or a disease or disorder related thereto,comprising administering to a mammal a therapeutically effective amountof a material selected from the group consisting of a PRODH comprisingan amino acid sequence of SEQ ID NO:2, a conservative variant thereof, afragment thereof, or an analog or derivative thereof. The phrase“therapeutically effective amount” is used herein to mean an amountsufficient to treat, and preferably decrease by at least about 30percent, more preferably by at least 50 percent, most preferably by atleast 90 percent, a symptom or symptoms associated with schizophrenia ora disease or disorder related thereto in a subject. In a particularembodiment, the PRODH, conservative variants thereof, fragments thereof,or analogs or derivatives thereof are delivered parenterally asdescribed above.

[0550] In yet another aspect of the present invention, methods oftreatment and manufacture of a medicament are provided. Conditionsalleviated or modulated by the administration of the present derivativesare those indicated above.

[0551] Dosages.

[0552] For all of the above molecules, as further studies are conducted,information will emerge regarding appropriate dosage levels fortreatment of various conditions in various patients, and the ordinaryskilled worker, considering the therapeutic context, age and generalhealth of the recipient, will be able to ascertain proper dosing.

Methods of Assaying Drugs or Agents Using Mice and the PPI Test

[0553] Disclosed infra is a new mouse strain useful in assaying drugs oragents for the ability to treat schizophrenia, and the teachings toproduce such a mouse. Furthermore, the PPI test which is used to examinethe mice is discussed in detail below.

[0554] Hence, the present extends to a method of identifying drugs oragents useful in treating schizophrenia or a disease or disorder relatedthereto comprising the steps of:

[0555] a) performing an first pre-pulse inhibition test (PPI) test on amouse having within its genome two copies of an isolated variant alleleof a Prodh gene comprising a DNA sequence of SEQ ID NO:7, wherein bothcopies are capable of expressing a mutant Prodh comprising an amino acidsequence of SEQ ID NO:8, to obtain a first percentage of inhibition ofstartle response;

[0556] b) administering the potential drug or agent to the mouse;

[0557] c) performing a second PPI test on the mouse to obtain a secondpercentage of inhibition of startle response; and

[0558] d) comparing the first percentage to the inhibition of startleresponse with the second percentage of startle response,

[0559] wherein an increase in percentage of inhibition in the secondpercentage of inhibition relative to the first percentage of inhibitionis indicative of the ability of the drug or agent to treat schizophreniaor a disease or disorder related thereto. Thus, if the percentage ofinhibition of startle response in the mouse having within its two activecopies of an isolated variant allele of a Prodh gene comp mouse afteradministration of the drug or agent is greater than the percentage ofinhibition of startle response in the Pro/Re mouse prior to inhibition,then the drug or agent has the ability to treat schizophrenia or adisease or disorder related thereto, such as obsessive compulsivedisorder (OCD), bipolar disorder (BP) or major depressive disorder.

[0560] Also, the present invention extends to a method for identifying adrug or agent for treating schizophrenia or a disease or disorderrelated thereto. An example of such a method comprises the steps of:

[0561] performing an first pre-pulse inhibition test (PPI) test on an F3generation mouse from a cross Pro/Re X C57B1/6J wild-type, wherein theF3 generation mouse has two copies within its genome of an isolatedvariant allele of a Prodh gene comprising a DNA sequence of SEQ ID NO:7which are capable of expressing a mutant Prodh comprising an amino acidsequence of SEQ ID NO:8, to obtain a first percentage of inhibition ofstartle response;

[0562] administering the potential drug or agent to the F3 generationmouse from a cross of Pro/Re X C57B1/6J wild-type;

[0563] performing a second PPI test on the F3 generation mouse from across of Pro/Re X C57B1/6J wild-type to obtain a second percentage ofinhibition of startle response; and comparing the first percentage tothe inhibition of startle response with the second percentage of startleresponse,

[0564] wherein an increase in percentage of inhibition in the secondpercentage of inhibition relative to the first percentage of inhibitionis indicative of the ability of the drug or agent to treat schizophreniaor a disease or disorder related thereto.

[0565] What's more, the present invention extends to a method foridentifying a drug or agent for use in treating schizophrenia or adisease or disorder related thereto, comprising the steps of:

[0566] administering the drug or agent to an F3 generation mouse from across of Pro/Re X C57B1/6J wild-type, wherein the F3 generation mousehas two copies within its genome of an isolated variant allele of aProdh gene comprising a DNA sequence of SEQ ID NO:7 which are capable ofexpressing a mutant Prodh comprising an amino acid sequence of SEQ IDNO:8;

[0567] performing a PPI test on the F3 generation mouse from a cross ofPro/Re X C57B1/6J wild-type to obtain a percentage of inhibition of thestartle response in the F3 generation mouse from a cross of Pro/Re XC57B1/6J wild-type which was administered the drug or agent; and

[0568] comparing the percentage of inhibition of the startle response inthe F3 generation mouse from a cross of Pro/Re X C57B1/6J wild-type withthe percentage of inhibition of the startle response in an unmedicatedF3 generation mouse from a cross of Pro/Re X C57B1/6J wild-type, whereinthe F3 generation mouse has two copies within its genome of an isolatedvariant allele of a Prodh gene comprising a DNA sequence of SEQ ID NO:7which are capable of expressing a mutant Prodh comprising an amino acidsequence of SEQ ID NO:8.

[0569] An increase in percentage of inhibition in the percentage ofinhibition in the medicated mouse relative to the percentage ofinhibition in the unmedicated mouse is indicative of the ability of thedrug or agent to treat schizophrenia or a disease or disorder relatedthereto.

[0570] Furthermore, the present invention extends to a method foridentifying a drug or agent for use in treating schizophrenia or adisease or disorder related thereto, comprising the steps of:

[0571] administering the drug or agent to a mouse having within itsgenome two copies of an isolated variant allele of a Prodh genecomprising a DNA sequence of SEQ ID NO:7, wherein both copies arecapable of expressing a mutant Prodh comprising an amino acid sequenceof SEQ ID NO:8;

[0572] performing a PPI test on the mouse to obtain a percentage ofinhibition of the startle response in the mouse; and

[0573] comparing the percentage of inhibition of the startle response inthe mouse with the percentage of inhibition of the startle response inan unmedicated mouse having within its genome two copies of an isolatedvariant allele of a Prodh gene comprising a DNA sequence of SEQ ID NO:7,wherein both copies are capable of expressing a mutant Prodh comprisingan amino acid sequence of SEQ ID NO:8.

[0574] An increase in percentage of inhibition in the percentage ofinhibition in the medicated mouse relative to the percentage ofinhibition in the unmedicated mouse is indicative of the ability of thedrug or agent to treat schizophrenia or a disease or disorder relatedthereto.

[0575] In addition, the present invention extends to a method foridentifying a drug or agent for use in treating schizophrenia or adisease or disorder related thereto, comprising the steps of:

[0576] administering the drug or agent to a mouse having within itsgenome two copies of an isolated variant allele of a Prodh genecomprising a DNA sequence of SEQ ID NO:7, wherein both copies arecapable of expressing a mutant Prodh comprising an amino acid sequenceof SEQ ID NO:8;

[0577] performing a PPI test on the mouse to obtain a percentage ofinhibition of the startle response in the mouse; and

[0578] comparing the percentage of inhibition of the startle response inthe mouse with the percentage of inhibition of the startle response inan unmedicated mouse having within its genome two copies of an isolatedProdh gene comprising a DNA sequence of SEQ ID NO:3, wherein both copiesare capable of expressing a Prodh comprising an amino acid sequence ofSEQ ID NO:4.

[0579] If the percentage of inhibition of the startle response in themedicated mouse is statistically equivalent to the percentage ofinhibition in the mouse capable of expressing Prodh comprising a DNAsequence of SEQ ID NO:4, then the drug or agent has the ability to treatschizophrenia or a disease or disorder related thereto.

[0580] In another embodiment, the present invention extends to an amethod for identifying a drug or agent for use in treating schizophreniaor a disease or disorder related thereto, comprising the steps of:

[0581] administering the drug or agent to an F3 generation mouse from across of Pro/Re X C57B1/6J wild-type, wherein the F3 generation mousehas two copies within its genome of an isolated variant allele of aProdh gene comprising a DNA sequence of SEQ ID NO:7 which are capable ofexpressing a mutant Prodh comprising an amino acid sequence of SEQ IDNO:8;

[0582] performing a PPI test on the F3 generation mouse from a cross ofPro/Re X C57B1/6J wild-type administered the drug or agent to obtain apercentage of inhibition of the startle response in the mouse; and

[0583] comparing the percentage of inhibition of the startle response inF3 generation mouse from a cross of Pro/Re X C57B1/6J wild-typeadministered the drug with the percentage of inhibition of the startleresponse in an F3 generation mouse from a cross of Pro/Re X C57B1/6Jwild-type, wherein the F3 generation mouse has two copies within itsgenome of an isolated Prodh gene comprising a DNA sequence of SEQ IDNO:3 which are capable of expressing a Prodh comprising an amino acidsequence of SEQ ID NO:4

[0584] If the percentage of inhibition of the startle response in themedicated mouse is statistically equivalent to the percentage ofinhibition in the mouse capable of expressing Prodh comprising a DNAsequence of SEQ ID NO:4, then the drug or agent has the ability to treatschizophrenia or a disease or disorder related thereto.

[0585] The PPI test involves the evaluation of sensimotor gating. Inparticular, this test permits the evaluation of sensormotor gating fromthe degree of inhibition of an acoustic startle response by a prepulsepreceding by 100 msec an abrupt startling stimulus (pre-pulseinhibition). PPI was recorded using a combination of two startle levels(100 and 115 dB) and two prepulse levels (82 and 90 dB) and is expressedas:

100-[(response to startle stimulus following pre-pulse/response tostartle stimulus alone)×100].

[0586] Testing was conducted in an SR lab system (San Diego Instruments)Each of two accoustically insulated startle chambers contained atransparent acrylic cylinder (4 cm in diameter) mounted on a frame towhich a motion sensor was attached for the detection and transduction ofmovement, and a sound generation system for the delivery of backgroundwhite noise and acoustic stimuli. Immediately after placement in thechamber, the animal was given a 5 min. Acclimation period during whichbackground noise (67 dB) was continually present, and then received 6sets of the following 7 trial types counterbalanced to control fororder: Trial 1: 40 ms, 100 dB noise burst alone; trial 2: 40 ms, 115 dBnoise burst alone; trial 3-6: 100 dB or 115 dB startle stimuli preceded100 ms by a 20 ms, 82 dB or 90 dB noise burst (prepulse); trial 7:no-stimulus/background noise alone (67 dB). Intertrial interval wasvariable (10-20 sec with an average of 15 sec). At the beginning of theblock of 42 trials, the animal received the following 3 trials: 1no-stimulus/background noise along (67 dB) trial, 1 startle stimulusalone trial for both 100 dB and 115 dB. At the end of the block of 42trials the animal received the same 3 trials again in reverse order.

[0587] The background noise level was 67 dB during the entire testingsession. Data was analyzed using ANOVA with repeated measures.

[0588] The present invention may be better understood by reference tothe following non-limiting Example, which is provided as exemplary ofthe invention. The following Example is presented in order to more fullyillustrate the preferred embodiments of the invention. It should in noway be construed, however, as limiting the broad scope of the invention.

EXAMPLE

[0589] Evidence for an association between schizophrenia susceptibilityand hemizygous deletions in chromosome 22q11 has previously beendetermined [Karayiorgou et al., Proc. Natl. Acad. Sci. U.S.A. 92, 7612(1995)]. More specifically, three hemizygous cryptic deletions at 22q11in a sample of 300 unrelated schizophrenic patients have previously beenreported and characterized. [M. Karayiorgou et al., Proc. Natl. Acad.Sci. U.S.A. 92, 7612 (1995); M. Karayiorgou et al., Amer. J. Med. Genet.74, 677 (1997)] The frequency of this microdeletion in the generalpopulation is estimated to be ^(˜)0.02% (although the latter is mostlikely an underestimate) and no deletions were found in a sample of 200healthy controls. [M. Karayiorgou et al., Proc. Natl. Acad. Sci. U.S.A.92, 7612 (1995); M. Karayiorgou et al., Amer. J. Med. Genet. 74, 677(1997)] The identified locus (^(˜)1.5 Mb in size) is located in theproximal part of a region at chromosome 12q11, and has been implicatedindependently in schizophrenia susceptibility through linkage studies.[Karayiorgou, M. and Gogos, J., Neuron 19, 967 (1997)] This locusoverlaps with a critical region involved in the etiology ofVelocardio-facial (VCFS)/DiGeorge (DGS) syndromes [Driscoll et al., J.Med. Genet. 30, 813 (1993)] It has been shown that ^(˜)29% VCFS childrenwith 22q11 deletions develop schizophrenia or schizoaffective disorderin adolescence and adulthood [Pulver, A. E., et al., J. Nerv. Ment. Dis.182, 476 (1994)] an estimate confirmed by a more recent independentstudy. 22q11 deletions have been identified among schizophrenia patientsof diverse ethnic origins (Chinese, Israeli, British, Danish) and onestudy implicated the 22q11 region in early-onset schizophrenia Yan etal., Am. J. Med. Genet. 81, 41 (1998)1. In addition, the increased ratesof comorbid obsessive compulsive disorder (OCD) or symptoms (OCS) amongschizophrenic patients with the 22q11 microdeletion locus [KarayiorgouM., Gogos, J A, et al., Genotype and Phenotype Analysis of the 22q11Schizophrenia Susceptibility Locus. Cold Spring Harbor Symposia onQuantitative biology, Vol. LXI, pp. 835-843, Cold Spring Harbor Press,Cold Spring Harbor, N.Y. (1996); Karayiorgou, M. et al., Proc. Natl.Acad. Sci. 94, 4572-4575 (1997)] and similarly increased rates ofanxiety, OCS and OCD in children and adults with the 22q11 microdeletionin the absence of schizophrenia indicates that the 22q11 genomic regionmay harbor one or more genes predisposing to schizophrenia or a diseaseor disorder related thereto.

[0590] Furthermore, an association in a recessive manner between a lowactivity allele of the Catechol-O-methyltransferase (COMT) gene, locatedat the proximal part of the 22q11 deleted region, and a susceptibilityto OCD, particularly in males, has been reported. [Karayiorgou et al.1997, supra.] The observation that ^(˜)20% of schizophrenia patientsreport obsessions and compulsions, features that are found in only 1-2%of the general population [Eisen, J. L & Rasmussen, S. A., ObsessiveCompulsive Disorder with Psychotic Features. J. Clin. Psychiatry54:373-379 (1993); Berman, I. Kalinowski, A., Berman, S. M., Lengua, J.,and Green, A. I., Obsessive and Compulsive Disorders in ChronicSchizophrenia. Compr. Psychiatry 36:6-10 (1995)1 indicates thatschizophrenia and OCD may share some pathophysiological and geneticcomponents. For example, one common central processing mechanism thatseems to be affected in patients with schizophrenia and OCD issensorimotor gating. Patients with schizophrenia and OCD demonstratepoor sensorimotor gating of the startle response as measured by impairedprepulse inhibition of an acoustic response and this may lead to sensoryoverload, distractibility and cognitive fragmentation.

[0591] Reported herein is the isolation and characterization of thehuman homolog (FIG. 1A) of the D. melanogaster slgA prolinedehydrogenase gene (PRODH), which is responsible for the behavioralphenotype of the D. melanogaster sluggish-A mutant [Hayward, D. C., etal., Proc. Natl. Acad. Sci. USA 90, 2979 (1993)]. The gene was localizedat the most centromeric part of the 22q11 deletions and was shown to beexpressed in several tissues, including brain. Mapping of PRODHindicates this gene may contribute to the psychiatric phenotypeassociated with the 22q11 deletions, because proline has long beensuspected to serve as a modulator of a synaptic transmission in themammalian brain and in addition, proline dehydrogenase is involved inthe biosynthesis and release of the neurotransmitter glutamate.

[0592] Elevated levels of proline have been reported in a DiGeorge/VCFSpatient [Jacken, J., Goemans, N. Frynes, J. -P, Francois, I., de Zegher,F., J. Inherit. Metab. Dis. 19. 275 (1996)], wherein DiGeorge/VCFS is acontiguous gene microdeletion syndrome involving chromosome 22q11. Thede novo origin of the deletion and the fact that both the parents andthe sister of the affected proband had normal proline levels, suggestedthat deletion of a gene located within the 22q11 region is responsiblefor the observed hyperprolinaemia. Proline dehydrogenase is the firstenzyme of proline catabolism that converts proline toΔ1-pyrroline-5-carboxylate [Wang, S. S. and Brandriss, M. C., Mol. CellBiol. 6, 2638 (1986); Wang, S. S. and Brandriss, M. C., Mol. Cell Biol.7, 4431 (1987)] and dysfunction of this enzyme is expected to result inabnormal proline metabolism. The Drosophila melanogaster sluggish-A gene(slgA) was previously shown to encode a proline dehydrogenase; activityof this enzyme is abnormally low in the slgA mutant that presentsbehavior abnormalities, and is restored to wild-type levels intransgenic flies carrying the wildtype slgA gene [Hayward, D. C., etal., Proc. Natl. Acad. Sci. USA 90, 2979 (1993)]. Database searchesusing the sequence of the D. melanogaster slgA gene identified severalhuman Expressed Sequence Tag (EST) cDNA clones, encoding a proteinsequence with a strong match to the D. melanogaster slgA coding region.In particular, an insert from EST ym93b08.r1 from a human brain library(GenBank R88591) was used to screen a human cerebellar as well as akidney cDNA library using a BLAST search provided by the NationalInstitutes of Health at http://www.ncbi.nlm.nih.gov/cgi-bin/BLAST on theWorld Wide Web. A score having an E value of 4e⁻⁰⁶ is considered a“strong match”. A cDNA fragment was used as a hybridization probeagainst a panel of monochromatic hybrids as well as an array of ninephage artificial chromosomes (PACs) previously mapped and ordered withinthe 22q11 region [Carlson, C. et al., Am. J. Hum. Genet. 61, 620(1997)]. Only chromosome 22 (not shown) and PAC-P457M14 provided apositive signal (FIG. 1B). This PAC is included in the smallest 22q11deletion identified to date in patients with schizophrenia [M.Karayiorgou et al., Proc. Natl. Acad. Sci. U.S.A. 92, 7612 (1995); M.Karayiorgou et al., Amer. J. Med. Genet. 74, 677 (1997)] suggesting thatthe human homologue of slgA maps within the deleted region. Mapping wasconfirmed by PCR analysis on a previously described [M. Karayiorgou etal., Proc. Natl. Acad. Sci. U.S.A. 92, 7612 (1995); M. Karayiorgou etal., Amer. J. Med. Genet. 74, 677 (1997)1 somatic cell hybrid linecarrying a copy of the deleted chromosome 22 (not shown). Libraryscreening, Northern blotting and hybridization, reverse transcriptionPCR and 5′ Rapid Amplification of cDNA ends (5′ RACE) were performedaccording to standard protocols [J. Sambrook, E. F. Fritsch, T.Maniatis, Molecular Cloning: A Laboratory Manual (Cold Spring HarborLaboratory Press, Cold Spring Harbor, N.Y., 2nd edition, 1989)]. Thesequence of both human and mouse clones was obtained by primer walkingfrom both strands. Sequence alignment and estimations of sequencesimilarity were performed using the program DNASTAR. 22q11 PAC cloneswere provided by P. deJong, Roswell Polytechnical College and Institute,Buffalo, N.Y. Monochromatic hybrids were purchased from BIOSLaboratories (New Haven, Conn.).

[0593]FIG. 1A shows an alignment of the predicted human PRODH with theD. melanogaster SLGA and yeast PUT1 proline dehydrogenase proteins, aswell as with sequence from the mouse homologue (see below). The humanand D. melanogavter proteins show 40.4% overall similarity as defined inthe “DNASTAR LASERGENE” software program (DNASTAR, Inc. 1228 South ParkStreet, Madison, Wis. 53715), although the region of highest similarityamong all homologues is localized toward the C-terminus where the humanand D. melanogaster proteins are 60.4% similar. Northern blot analysisand autoradiography revealed a 2.4-2.5 kb band in several human tissues(such as heart, lung, liver, skeletal muscle, kidney, pancreas),including brain (not shown) and higher resolution analysis of the brainexpression pattern revealed widespread distribution of the mRNA inseveral regions (FIG. 1C).

[0594] To determine whether the PRODH gene contributes to psychiatricphenotypes associated with the 22q11 deletions, two complementarystudies were performed and reported herein. The first study involvesisolation of the mouse Prodh gene, and identification of a mutation ofthis gene in the Pro/Re hypeprolinemic mouse strain. Furthermore,analysis of the startle response attenuation by prepulse inhibition(PPI) in these mice was used as a measure of sensorimotor gating, acentral processing mechanism that is affected in patients withschizophrenia and OCD. The second study involved identification of PRODHgene variants that can be used to examine their inheritance in nuclearfamilies with schizophrenia and obsessive compulsive disorder. Thesestudies are described in more detail below.

[0595] In the first study reported herein, human PRODH cDNA was used asa probe to isolate and sequence mouse Prodh cDNA from a cerebellar cDNAlibrary. The mouse and human proteins show 86% similarity (FIG. 1A).Northern blot analysis and autoradiography revealed a 2.4-2.5 kb band inseveral mouse tissues (such as heart, lung, liver, skeletal muscle,kidney, pancreas) including brain (FIG. 2A). The PRO/Re mouse [R. L.Blake and E. S. Russell, Science 176, 809 (1972)], wherein the PRO/Restrain is a highly inbred strain developed by over 25 generation siblinginbreeding starting from an original cross between 129/ReJ and C57BL/6Jstrains, carries a presumably homozygous defect in proline dehydrogenaseactivity. In order to understand the contribution of the PRODH gene inthe 22q11 associated psychiatric phenotype, the mouse Prodh gene fromthe PRO/Re mice was isolated, and a G to T substitution was identified135 nucleotides 5′ of the native termination codon has been identifiedthat results in a premature translational termination (FIG. 1A, FIG.2B). This substitution eliminates an MnlI site, and this information wasused for genotyping the F3 generation from a cross between the originalPro/Re and C57/B6 wild type strain (F2 heterozygotes were intercrossedand offspring were genotyped by PCR]. Analysis of plasma amino acidlevels demonstrated that presence of the identified mutation correlateswith increased levels of proline in F3 mice, and, thus most likelyaccounts for the hyperprolinemia in the Pro/Re Strain. Specifically, F3homozygous for the mutation present an average seven fold increase ofthe blood proline levels. In contrast, comparisons of glutamate levelsdid not reveal any significant changes (FIG. 2C).

[0596] Adult Prodh-deficient homozygous mice (TAG/TAG) were normal inappearance and development and had normal viability. However, 2-3 monthold male Prodh-deficient homozygous mice weighed ^(˜)10% less than wildtype littermates (GAG/GAG) [F(1.33)=12.625, p=0. 0012)].

[0597] Brain morphology appeared identical in homozygous mice and theirwild type littermates by gross evaluation. Upon histological examinationof sections, cell groups in the forebrain and diencephalon appeared tobe well formed with no obvious neuroanatomical alterations. Abnormalproline metabolism may alter biosynthesis and release of glutamate andy-aminobutyric acid (GABA) [Johnson, J. L. and Roberts, E., Brain Res.323, 247 (1984); Yoneda, Y. and Roberts, E., Brain Res. 239, 479 1982);Yoneda, Y., Roberts, E., and Dietz, G. W., J. Neurochem. 38, 1686(1982)]. Because L-glutamate is the principal neuromuscular transmitterin D. melanogaster, it has been hypothesized that a deficiency inproline dehydrogenase may alter L-glutamate metabolism in specific nervecell populations in D. melanogaster, thus being responsible for thesluggish phenotype reported by Hayward et al., (1986). GABA is thoughtto be the major inhibitory neurotransmitter of various inhibitoryinterneurons. Glutamate, in addition to being a major excitatory aminoacid transmitter, may also serve as a precursor of GABA and aspartate,another excitatory transmitter [Johnson, et al (1984). Therefore, basallevels of glutamate, GABA and aspartate were compared in extracts fromfrontal cortex, hypothalamus, amygdala, and hippocampus of homozygousmutant and wild type animals (FIG. 2D) [Luine, V. N., Grattan, D. R.,and Selmanoff, M., Brain Res. 747, 165 (1997); Grattan, D. andSelmanoff, M., J. Neurochem. 60, 2254 (1993)]. The frontal cortexsection taken was between approximately +3.08 and +1.70 mm (according tothe atlas of K. B. J. Franklin and G. Paxinos, The Mouse Brain inStereotaxic Coordinates, Academic Press, New York, 1997) and twodorsomedial punches were taken. It contained the medial and ventralorbital cortex and the pre- and infra-limbic cortex. One punch of thedorsal hippocampus was taken between approximately −2.0 and −3.0 mm. Thehypothalamus was sampled with bilateral punches between approximately0.34 and 2.18 mm and consisted of the dorso and ventromedial nuclei,anterior hypothalamic nucleus, and arcuate nucleus. Aminoacidsglutamate, aspartate, glycine, serine and γ-aminobutyric acid (GABA)- inthe brain samples were measured using HPLC with electrochemicaldetection following precolumn derivatization of the sample with0-phthalaldehyde and beta-mercaptoethanol. Briefly, punched tissues wereexpelled into sodium acetate buffer, pH 5.0, containing approximately 25ng of homoserine as an internal standard (150 μl for cortex and 200 μlfor hippocampus). The samples were centrifuged and 15 μl of supernatant,following automated precolumn derivatization, was injected using arefrigerated, Waters 717 Autosampler onto a Brownlee Velosep RP-18 3micron column. Amino acids were detected with an ESA model 5200Coulochem II detector with a model 5011 analytical cell with electrodesset at +0.1 mV to oxidize and remove derivatization contaminants and at+0.45 mV to oxidize and detect derivatized amino acids. Details of themobile phase and derivatization reagent can be found in Grattan andSelmanoff, 1993. Picograms of amino acids were calculated with theWaters Millenium computer system by comparing the ratio of the peakheights of the transmitters and the internal standard in samplechromatograms with ratio of peak heights in chromatograms from externalamino acid standards. Proteins were determined in the sample pellets andconcentrations of amino acids are expressed as ng/ug of protein.Measurements were made in a single cohort of mice consisting of male andfemale homozygous (n=15) and wild type (n=1).

[0598] An 8% decrease in the levels of glutamate was observed in thefrontal cortex of the Prodh-deficient mice but this did not reachstatistical significance (90.7±5.6 versus 98.8±10.1, p=0.58). However, asignificant ⁻25% decrease of GABA levels (5.7±0.3 versus 7.7±0.6,p=0.03) and a ⁻18% decrease of the aspartate levels (9.5±0.5 versus11.5±0.4, p=0.01) was observed in the same brain region. Similarly,significant decreases in the levels of glutamate (⁻13%, 36.9±1.0 verses42.3±1.4, p=0.0062), GABA (^(˜)10%, 9.3±0.1 verses 10.4±0.4, p=0.01) andaspartate (⁻10%, 8.0±0.2 verses 8.9±0.3, p=0.03) were observed in thehypothalamus of homozygous mutants. By contrast, no significantdifferences were observed among animals of either genotype in the levelsof these three neurotransmitters in the amygdala and hippocampus. Inaddition, no significant differences were observed in the levels ofglycine (another inhibitory neurotransmitter), in all brain regionsexamined.

[0599] Homozygous mutant mice and wild type littermates, 2-3 months oldat the onset of testing were analyzed in behavioral assays of sensoryreception/processing, locomotion and anxiety. Forty two homozygous and26 wild type littermate mice of both sexes, 2-3 months old at the onsetof testing were used for the behavioral analysis. Animals were housed ingroups of 2-4 and were maintained on a reverse 12:12 h light-dark cyclewith lights off at 0700 hrs. All testing occurred between 0900 and 1800hrs. Prior to all testing, animals were handled and weighed. Thebehavioral tests were performed in the following order: locomotoractivity, light/dark transition, PPI and habituation of the startleresponse. At the conclusion of all behavioral assays the genotypes ofall mice were reconfirmed by PCR analysis. One central processingmechanism that is affected in patients with psychiatric disorders(primarily schizophrenia, and probably OCD) is sensorimotor gating, aneural filtering process that allows attention to be focused on a givenstimulus. This impaired attentional filtering is thought to result insensory overload, distractibility, cognitive fragmentation and possiblypsychotic symptoms [D. Braff et al., Psychophysiology 15, 339 (1978); C.Grillon, R. Ameli, D. S. Charney, Biol. Psychiatry 32, 939 (1992); N. R.Swerdlow, C. H. Benbow, S. Zisook, Biol. Psychiatry 33, 298 (1993); W.Perry and D. L. Braff, Am. J. Psychiatry 151, 363-367 (1994)1.Sensorimotor gating can be evaluated from the degree of inhibition of anacoustic startle response by a weak prepulse preceding by 30-500 msec anabrupt startling stimulus (FIG. 3A, pre-pulse inhibition, (PPI) [R.Paylor and J. N. Crawley, Psychopharmacology 132, 169 (1997); N. R.Swerdlow, D. L. Braff, N. Taaid, M. A. Geyer, Arch. Gen. Psychiatry 51,139 (1994); S. F. Logue, E. H. Owen, D. L. Rasmussen, J. M. Wehner,Neuroscience 80, 1075 (1997); A. E. Bullock, B. S. Slobe, V. Vasquez, A.C. Collins, Behav Neurosci. 111, 1353 (1997)]. PPI is one of a fewneuropsychological measures in which humans and rodents can be evaluatedmore or less in a similar fashion. In rodents, the neural circuitunderlying the startle reflex is thought to involve four to fivesynapses [M. Davis, D. S. Gendelman, M. D. Tischler, P. M. Gendelman, J.Neurosci. 2, 791 (1982)] and a number of centers and neurotransmittersmay exert prepulse influences on its function [M. A. Geyer and D. L.Braff, Schizophr. Bull. 13, 643 (1987)]. In addition, it has beensuggested that GABA-mediated presynaptic control of excitatoryneurotransmitter release may be involved in the control of gating ofsensory responses [Duter and R. A. Nicoll, Neuron 1, 585 (1988)] andGABAergic circuitry may partly mediate the decrease in sensorimotorgating induced by dopamine overactivity [N. R. Swerdlow, D. L. Braff, M.A. Geyer, Brain Res. 532, 146 (1990); M. H. Kodsi and N. R. Swerdlow,Brain Res. Bull. 43, 219 (1997)] and activation of nicotinic receptors[N. R. Swerdlow, D. L. Braff, M. A. Geyer, Brain Res. 532, 146 (1990);M. H. Kodsi and N. R. Swerdlow, Brain Res. Bull. 43, 219 (1997)].

[0600] Another measure of sensorimotor gating is based onelectoencephalographic techniques designed to study response to pairedauditory stimuli. In this case, suppression of the P50 wave in humansand the N40 wave in rodents by a second stimulus, when two auditorystimuli are presented in sequence, is used as a measure of normal gating[R. Freedman et al., in Schizophrenia: Origins, processes, treatment,and outcome, R. L. Cromwell and C. R. Snyder, Eds., (Oxford Univ. Press,New York, 1993), pp. 98-108; K. E. Stevens et al.,Neuropsychopharmacology 15, 152 (1996)]. Patients with schizophrenia aswell as some of their non-symptomatic family members show decreasedattenuation of the second P50 wave. Recently a genetic linkage has beendescribed between a DNA marker on chromosome 15 and decreased P50attenuation in some families with schizophrenia [R. Freedman et al.,Proc. Natl. Acad. Sci. U.S.A. 94, 587 (1997)].

Methods and Materials Library Screening

[0601] Library screening, Northern blotting and hybridization, reversetranscription PCR and 5′ Rapid Amplification of cDNA ends (5′ RACE) wereperformed according to standard protocols [J. Sambrook, E. F. Fritsch,T. Maniatis, Molecular Cloning: A Laboratory Manual (Cold Spring HarborLaboratory Press, Cold Spring Harbor, N.Y., 2nd edition, 1989)]. Thesequence of both human and mouse clones was obtained by primer walkingfrom both strands. Sequence alignment and estimations of sequencesimilarity were performed using the program DNASTAR. 22q11 PAC cloneswere provided by P. deJong, Roswell Polytechnical College and Institute,Buffalo, N.Y. Monochromatic hybrids were purchased from BIOSLaboratories (New Haven, Conn.).

Genotyping Mutants and Wild Type Littermates

[0602] Pro/Re and C57/B6 mice were purchased from Jackson Laboratories.For PCR genotyping for the identified mutation, the following primerswere used:

[0603] F: 5′GACCAAATCAGCTTCCCACT (SEQ ID NO:5);

[0604] R: 5′CCCTTCATGATGCTGCTGTT (SEQ ID NO:6).

[0605] Target sequences were amplified in a 50 Al reaction mixturecontaining 100 ng of genomic DNA in 50 mM KCl, 10 mM Tris-HCl (pH 8.3),1.5 mM MgCl₂, 0.1% gelatin, 50 mm of each primer, 200 mM of each dNTP(dATP, dCTP, dGTP, dTTP), and 1.5 u Taq polymerase. PCR was performed ina programmable PCR apparatus (MJ Research, Inc.). Amplification was asfollows: 80° C.×8 min (1×), 92° C.×1 min/60° C. ×40 sec/72° C.×1 min(30×), 72° C. ×7 min (1×), 4° C.×5 min. 45 μl of the amplified productwas digested with 20-40 μ of MnlI in a 60 μl reaction volume, accordingto the manufacturer's specifications. The digested product waselectrophoresed in a 4% 1× TBE NuSieve agarose gel.

[0606] Because of the development of a blood-brain barrier towardsproline early in development [J. L. Purdy and S. C. Bondy, Neuroscience1, 125 (1976)], plasma levels may not be adequately describing brainlevels, although previous analysis of a CD-1×Pro/Re F3 cross revealed anincrease in the brain proline levels as well [C. F. Baxter, R. A.Baldwin, J. L. Davis, J. J. Flood, Pharmac. Biochem. & Behav. 22, 1053(1985)]. The normal concentration of proline in human plasma is between100 and 450 mM. The concentration of proline in cerebrospinal fluid isconsiderably lower (4.2 mM) [C. R. Scriver and L. E. Rosenberg,Aminoacid metabolism and its disorders (Saunders, Philadelphia, 1973)].

[0607] The nucleotide sequence obtained from the cerebellar cDNA clonesoverlapped partly with the sequence of the kidney cDNA clones (data notshown). Additional sequence was obtained using a 5′RACE approach ontotal human brain mRNA. As part of a D. melanogaster/human sequencingproject, Campbell and colleagues [H. D. Campbell, G. C. Webb, I. G.Young, Hum. Genet. 101, 69 (1997)] also identified a human cDNA withhomology to slgA (the gene product was assigned the name PRODH). Thereported sequence is identical to the one determined in this study withthe exception of a G insertion in the 3′ UTR, 1793 base pairs downstreamof the initiator methionine. Analysis in a small number of individualssuggests that this variation represents either a sequencing artifact ora very rare polymorphism. In addition, homologues from (intentionallyleft blank) C. elegans [GeneBank Z81060, Sulston et al., Nature 356, 37(1992)] and Arabidopsis thaliana ]N. Verbruggen, X. -J. Hua, M. May, M.Montagu, Proc. Natl. Acad. Sci. U.S.A. 93, 8787 (1996)] have also beenreported.

Testing of Prodh-Deficient Mice and Wild Type Littermates

[0608] Forty two homozygous and 26 wild type littermate mice of bothsexes, 2-3 months old at the onset of testing were used for thebehavioral analysis. Animals were housed in groups of 2-4 and weremaintained on a reverse 12:12 h light-dark cycle with lights off at 0700hrs. All testing occurred between 0900 and 1800 hrs. Prior to alltesting, animals were handled and weighed. The behavioral tests wereperformed in the following order: locomotor activity, light/darktransition, PPI and habituation of the startle response. At theconclusion of all behavioral assays the genotypes of all mice werereconfirmed by PCR analysis.

[0609] Spontaneous locomotor activity was tested in an open fieldapparatus, a clear acrylic chamber (40.5×40.5×30 cm) equipped withinfrared sensors for the automatic recording of horizontal activity(Digiscan Model RXYZCM, Accuscan Instruments, Inc., Columbus, Ohio).Each subject was initially placed in the center of the chamber and totaldistance traveled over the next 15 min was used as the measure ofactivity. To minimize the influence of anxiety on activity level,activity was monitored under indirect, very dim light andsound-attenuated conditions.

[0610] Following the initial test of locomotor activity, the effect ofthe mProdh mutation on anxiety-like behaviors (collectively termedanxiety reactivity or emotionality) was recorded in a dark/lightexploratory model in a 2-compartment light/dark box. The apparatus andconditions were similar to those used for the locomotor assay, exceptthat an enclosed black acrylic box (40×20.5×20.5 cm) was inserted intothe right half of the chamber with an opening (13×5 cm) allowing forfree passage between the two compartments monitored by an infrared beam.The open compartment was now directly illuminated by a 60-watt bulbplaced 60 cm above the floor of the compartment. Animals were initiallyplaced in the dark compartment and data collection commenced immediatelyfor 10 min. Previous work assessing the effects of anxiolytic andanxiogenic agents has established the validity of this behavioralprocedure in evaluating anxiety-like behaviors in rodents. Variablesrecorded as a measure of anxiety included latency to emerge from thedark compartment into the more aversive brightly lit compartment, andamount of time spent ambulating in each of the two compartments.

[0611] Testing was conducted in a SR-Lab system (San Diego Instruments,San Diego, Calif.). Each of two acoustically insulated startle chamberscontained a transparent acrylic cylinder (4 cm in diameter) mounted on aframe to which a motion sensor was attached for the detection andtransduction of movement, and a sound generation system for the deliveryof background white noise and acoustic stimuli. To ensure comparablesensitivities of these detectors across the two chambers, a vibratingstandardization unit (San Diego Instruments, San Diego, Calif.) wasused. A CompuAdd 386 microprocessor and SDI interface board and softwarewere used for the delivery of stimuli and response recording (100 1-msreadings beginning at startle stimulus onset). Response amplitude wascalculated as the maximum response level occurring during the 100 msrecording. Both chambers were calibrated for equivalent stimulusintensities and response sensitivities with a digital sound level meter(RadioShack), and experimental groups were balanced across chambers.Because animals can in principle habituate to the prepulse [J. C.Gewirtz and M. Davis, Behav. Neurosci. 109, 388 (1995)], as well as tothe startle stimulus [T. D. Blumenthal, Psychophysiology 34, 446(1997)], the number of trials was kept to the essential minimum. Thechoice of the two prepulse bursts used in this experiment was based on apilot experiment. Immediately after placement in the chamber, the animalwas given a 5 min acclimation period during which background noise (67dB) was continually present, and then received 6 sets of the following 7trial types counterbalanced to control for order: Trial 1: 40 ms, 100 dBnoise burst alone; Trial 2: 40 ms, 115 dB noise burst alone; Trial 3-6:100 dB or 115 dB startle stimuli preceded 100 ms by a 20 ms, 82 dB or 90dB noise burst (prepulse); Trial 7: no-stimulus/background noise alone(67 dB). Intertrial interval was variable (10-20 sec with an average of15 sec). At the beginning of the block of 42 trials, the animal receivedthe following 3 trials: 1 no-stimulus/background noise alone (67 dB)trial, 1 startle stimulus alone trial for both 100 dB and 115 dB. At theend of the block of 42 trials the animal received the same 3 trialsagain in reverse order. The background noise level was 67 dB during theentire testing session. Data were analyzed using ANOVA with repeatedmeasures.

[0612] In order to obtain additional information on the perception ofthe employed prepulse stimuli by wild type and mutant mice, one weekafter the completion of the PPI experiment, prepulse alone startleresponses were evaluated at 82 dB and 90 dB. Mice were exposed to threeconsecutive 40 ms stimuli: 67 dB white noise (background trial), 82 dB(prepulse 1 trial), and 90 dB (prepulse 2 trial), distributedpseudorandomly and separated by an average of 15 sec intertrialintervals. This sequence was repeated 10 times, that is, each stimuluswere presented 30 times. The background noise level was 67 dB during theentire testing session. Whereas a prepulse alone startle response to 82dB was barely detectable over background for both genotypes, a responseto 90 dB could be reliably evaluated [there was a significant differencebetween response levels to 67 dB and 90 dB for both wild type (p=0.0017)and homozygous mutant animals (p=0.0001)]. The amplitude of the startleresponse at 90 dB was ^(˜)20% of the one observed for the 100 dB startlestimulus and it was indistinguishable between the two genotypes (p=0.6).Induction of a startle response by the 90 dB prepulse stimulus isunlikely to interfere with the interpretation of the observed PPIpatterns since previous extensive analysis of different mouse strainsdemonstrated that no correlation exists between the startle response to90 dB stimulus and the level of prepulse inhibition obtained with a 90dB prepulse. Moreover, the same analysis showed that there is nocorrelation between the strain-specific dB threshold for induction of astartle response and the level for prepulse inhibition with any of theprepulse stimuli.

[0613] Habituation of the acoustic startle response was evaluated asfollows: 36 homozygous mutant and 20 wild type animals were exposed tostartle stimuli of broadband 115 dB for 40 ms with an average of 15 sec(10-20 sec) intertrial interval, in the presence of 67 dB broadbandbackground noise. The session began with 5 min acclimation period,followed by 5 consecutive administrations of the 40 ms broad-band 115 dBburst. This block was repeated 24 times, resulting to 120 consecutivepresentations of the 115 dB noise. Weight-corrected startle magnitudevalues (Vmax) were averaged for each block, expressed as percentage ofthe first block and analyzed using ANOVA with repeated measures. Inaddition, slopes of the habituation curves were analyzed by simplelinear regression analysis.

Results and Conclusion

[0614] Prodh-deficient mice and wild type littermates were examined forabnormalities in sensorimotor gating. PPI was recorded, using acombination of two startle levels and two prepulse levels and expressedas 100-[(response to startle stimulus following pre-pulse/response tostartle stimulus alone)×100] (such that higher percentages representgreater levels of inhibition, FIG. 3A). This experiment demonstrated asignificant attenuation in the overall level of PPI in the homozygousmutant mice compared to wild type littermates [F(1,66)=6.14, p=0.015](FIG. 3B). No sex differences were noted (p=0.866) and the % PPIincreased with increasing prepulse levels in both genotypes. Twofeatures of the startle responses were also evaluated: amplitude of thestartle response (ASR) and habituation (the decrement in the ASR overrepeated presentations of the same stimulus, a measure of plasticityexhibited by startle responses). ASR can be affected by several factors,including the integrity and excitability of the responding neurons insubcortical auditory centers [J. F. Willot, J. Kulig, T. Satterfield,Hear. Res. 16, 161 (1984); M. Koch, C. Kling, C. M. Becker, Neuroreport7, 806, 1996]. Analysis by genotype did not reveal significantdifferences in the weight-corrected startle amplitudes between wild typeand Prodh-deficient mice (FIG. 3C). Although a number of studies seem tosuggest a dissociation between startle amplitude and PPI [R. Paylor andJ. N. Crawley, Psychopharmacology 132, 169 (1997); V. P. Bakshi, N. R.Swerdlow, M. A. Geyer, J. Pharmacol. Exp. Ther. 271, 787 (1994); C.Johansson, D. M. Jackson, J. Zhang, L. Svensson, Pharmacol. Biochem.Behav. 52, 649 (1995)], this result indicates that the observedattenuation of PPI is not associated with an impaired startle responseand in addition reveals an anatomical and functional integrity of theresponsive neurons in the PRODH-deficient mice. Furthermore, homozygousmutant mice did not demonstrate any impairment in the habituation of theacoustic startle, at least under the conditions tested (FIG. 3D) [C. J.Wilson and P. M. Groves, J. Comp. & Physiol. Psychiatry 83, 492 (1973);M. A. Geyer and D. L. Braff, Psychophysiology 19, 1 (1982)]. Locomotoractivity and anxiety (reactivity) were also assayed. In sharp contrastto the observed sensorimotor gating deficits, no significant differencesin the total distance traveled, stereotypic behavior, or time spent inmargin versus center (an estimate of anxiety) were observed amonganimals of either genotype in the open field locomotor assay (FIG. 4A)[W. E. Crusio, H. Schwegler, J. H. van Abeelen, Behav. Brain Res. 32, 80(1989)]. In addition, no significant effect of genotype on latency toemerge into the more aversive brightly lit compartment, or time spentambulating in either light or dark compartment was observed in thedark-light assay for anxiety (FIG. 4B) [J. Crawley and F. K. Goodwin,Pharmacol. Biochem. Behav. 13, 167 (1980); C. Mathis, S. M. Paul, J. N.Crawley, Behav. Genet. 24, 171 (1994); C. Mathis, P. E. Neumann, H.Gershenfeld, S. M. Paul, J. N. Crawley, Behav. Genet. 25, 557 (1995)].

[0615] Elevated proline concentration has been previously associatedwith behavioral deficits in D. melanogaster (defective phototaxis andlow locomotor activity with indecisive movement pattern). The markedbehavioral abnormalities observed in the slgA mutant flies correlatedwith a twofold elevation of proline in the brain [D. C. Hayward et al.,Proc. Natl. Acad. Sci. U.S.A. 90, 2979 (1993)], suggesting thatbehavioral effects may not necessarily depend on grossly elevatedproline levels. In humans, PRODH deficiency is likely to be associatedwith hyperprolinemia type I, a rare metabolic disorder [J. M. Phang, G.C. Yeh, C. R. Scriver, in The Metabolic and Molecular Bases of InheritedDisease, C. R. Scriver, A. L. Beaudet, W. S. Sly, D. Valle, Eds.(McGraw-Hill, N.Y., 1995), pp. 1125-1146). Unlike most metabolicphenotypes, hyperprolinemia type I heterozygotes (such as patients withhemizygous 22q11 microdeletions) can demonstrate intermediate levelincreases of plasma proline [J. Jacken, N. Goemans, J. -P. Fryns, I.Francois, F. de Zegher, J. Inherit. Metab. Dis. 19, 275 (1996); Phang,et al., (1995)]. The nature of the effects of abnormally high proline(as in hyperprolinemia type I homozygotes) remain controversial and anexact link between the defect in proline metabolism and abehavioral/neurological phenotype in humans is still lacking. Several ofthe initially proposed features of hyperprolinemia type I (such aslearning deficits) have not been observed reproducibly even amonghyperprolinemic siblings of affected probands [F. Mollica and L.Provone, Acta Paediatr. Scand. 65, 206 (1976)]. Although under noobligation to explain such results, and certainly not intending to bebound by any hypothesis to explain these results, it is possible thatfortuitous clinical findings, allelic heterogeneity and geneticbackground-dependent penetrance account for the variable observations.The rarity of this disorder and the phenotypic inconsistencies haveimpeded systematic psychiatric evaluation of these patients, althoughthree cases of patients with psychiatric symptoms (schizophrenia,psychotic symptoms, autism) and increased blood proline levels have beenreported [T. L. Perry, J. W. Wright, S. Hansen, Biol. Psychiatry 18, 89(1983); M. Efron, N. Engl. J. Med. 272, 1243 (1965); T. Rokkones and A.C. Løken, Acta Paediatr. Scand. 57, 225 (1968)]. In mice, initialanalysis of the original Pro/Re strain, revealed specific learningdeficits J. L. Davis, R. M. Pico, J. F. Flood, Behav. Neur. Biol. 48,128(1987)] and studies in chicks have shown that intracerebraladministration of proline may result in selective disruption of memoryformation [A. Cherkin, M. J. Eckardt, L. K. Gerbrandt, Science 193, 242(1976)].

Identification and Characterization of Variant Alleles of the HumanProline Dehydrogenase Gene

[0616] In the second study reported herein, common sequence variationsin the more conserved C-terminal part of the human cDNA were scanned inninety-two individuals affected with schizophrenia. Genomic PCR andprimarily reverse transcription (RT) PCR using total mRNA isolated fromtransformed lymphocytes was applied. Eight polymorphisms were identifiedin this region:

[0617] a silent G to A transition at the third position of codon 83,which introduces a PstI site;

[0618] a C to T transition at the first position of codon 101 whichresults in a substitution of arginine for tryptophan;

[0619] a G to A transition at the second position of codon 101 whichresults in a substitution of arginine for glutamine;

[0620] a silent C to T transition at the first position of codon 247;

[0621] a C to T transition in the third position of codon 342;

[0622] a C to T transition in the third position of codon 421;

[0623] an A to G transition at the second position of codon 437, whichresults in a substitution of glycine for arginine; and

[0624] a silent T to C transition at the first position of codon 497,which introduces a PvuII site.

[0625] Experiments using RT-PCR with primers flanking introns, showedthat all variations were present in PRODH mRNA. Analysis of thesevariations in more extended samples revealed that the PvuII polymorphismis relatively frequent in that ⁻30% of chromosomes in a sample ofpatients with schizophrenia displayed this variation.

[0626] Moreover, statistical data from particular families whose memberssuffer from schizophrenia have been gathered, and demonstrate a directlink between the PRODH gene polymorphism comprising the silent T to Ctransition at the first position of codon 497, which introduces a PvuIIsite, and susceptibility of a subject to schizophrenia. In particular, atransmission disequilibrium test (TDT) [Spielman, R. S. et al.Transmission test for linkage disequilibirium: the insulin gene regionand insulin-dependent diabetes mellitus (IDDM). Am. J. Hum. Genet.(1993), 53:506-516] was performed on data from families in which memberssuffered from schizophrenia. This test takes affected individuals withtheir two parents. All three are typed for a marker, one allele of whichis believed to be associated with the disease. The TDT considers thecases where at least one parent is heterozygous at the marker allele(M1) which is suspected of being associated with the disease. One of thetwo marker alleles of each heterozygous patent is transmitted to eachaffected offspring and one is not. The test compares the frequency of M1among the transmitted and nontransmitted alleles. The significance ofthe association is tested by a simple X² test (Table 1). TABLE 1 Teststo determine whether marker allele M1 is associated with a disease. TDTtest Nontransmitted allele Transmitted allele M1 Not M1 M1 a b not M1 cd

[0627] The results of the TDT, set forth in FIG. 5, show there is astatistically significant preferential transmission of the PRODH genepolymorphism comprising the silent T to C transition at the firstposition of codon 497, which introduces a PvuII site, and susceptibilityof a subject to schizophrenia, or disease or disorders related thereto,such as obsessive compulsive disorder (OCD), bipolar disorder (BP), ormajor depressive disorder (MDD).

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[0714] The present invention is not to be limited in scope by thespecific embodiments describe herein. Indeed, various modifications ofthe invention in addition to those described herein will become apparentto those skilled in the art from the foregoing description and theaccompanying figures. Such modifications are intended to fall within thescope of the appended claims.

[0715] It is further to be understood that all base sizes or amino acidsizes, and all molecular weight or molecular mass values, given fornucleic acids or polypeptides are approximate, and are provided fordescription.

[0716] Various publications are cited herein, the disclosures of whichare incorporated by reference in their entireties.

1 9 1 1551 DNA Homo sapiens 1 atgctggaat ttgtgatgag agagtggaaaaaatccagga aacttctagg acagaggcta 60 ttcaacaagc tcatgaagat gaccttctatgggcattttg tagccgggga ggaccaggag 120 tccatccagc ccctgcttcg gcactacagggccttcggtg tcagcgccat cctggactat 180 ggagtggagg aggacctgag ccccgaggaggcagagcaca aggagatgga gtcctgcacc 240 tcagctgcgg agagggatgg cagtggcacgaataagcggg acaagcaata ccaggcccac 300 cgggccttcg gggaccgcag gaatggtgtcatcagtgccc gcacctactt ctacgccaat 360 gaggccaagt gcgacagcca catggagacattcttgcgct gcatcgaagc ctcaggtaga 420 gtcagcgatg acggcttcat agccattaagctcacagcac tggggagacc ccagtttctg 480 ctgcagttct cagaggtgct ggccaagtggaggtgcttct ttcaccaaat ggctgtggag 540 caagggcagg cgggcctggc tgccatggacaccaagctgg aggtggcggt gctgcaggaa 600 agtgtcgcaa agttgggcat cgcatccagggctgagattg aggactggtt cacggcagag 660 accctgggag tgtctggcac catggacctgctggactgga gcagcctcat cgacagcagg 720 accaagctgt ccaagcacct ggtagtccccaacgcacaga caggacagct ggagcccctg 780 ctgtcccggt tcactgagga ggaggagctacagatgacca ggatgctaca gcggatggat 840 gtcctggcca agaaagccac agagatgggcgtgcggctga tggtggatgc cgagcagacc 900 tacttccagc cggccatcag ccgcctgacgctggagatgc agcggaagtt caatgtggag 960 aagccgctca tcttcaacac ataccagtgctacctcaagg atgcctatga caatgtgacc 1020 ctggacgtgg agctggctcg ccgtgagggctggtgttttg gggccaagct ggtgcggggc 1080 gcatacctgg cccaggagcg agcccgtgcggcagagatcg gctatgagga ccccatcaac 1140 cccacgtacg aggccaccaa cgccatgtaccacaggtgcc tggactacgt gttggaggag 1200 ctgaagcaca acgccaaggc caaggtgatggtggcctccc acaatgagga cacagtgcgc 1260 ttcgcactgc gcaggatgga ggagctgggcctgcatcctg ctgaccacca ggtgtacttt 1320 ggacagctgc taggcatgtg tgaccagatcagcttcccgc tgggccaggc cggctacccc 1380 gtgtacaagt acgtgcccta tggccccgtgatggaggtgc tgccctactt gtcccgccgt 1440 gccctggaga acagcagcct catgaagggcacccatcggg agcggcagtt gctgtggctg 1500 gagctcttga ggcggctccg aactggcaacctcttccatc gccctgccta g 1551 2 516 PRT Homo sapiens 2 Met Leu Glu PheVal Met Arg Glu Trp Lys Lys Ser Arg Lys Leu Leu 1 5 10 15 Gly Gln ArgLeu Phe Asn Lys Leu Met Lys Met Thr Phe Tyr Gly His 20 25 30 Phe Val AlaGly Glu Asp Gln Glu Ser Ile Gln Pro Leu Leu Arg His 35 40 45 Tyr Arg AlaPhe Gly Val Ser Ala Ile Leu Asp Tyr Gly Val Glu Glu 50 55 60 Asp Leu SerPro Glu Glu Ala Glu His Lys Glu Met Glu Ser Cys Thr 65 70 75 80 Ser AlaAla Glu Arg Asp Gly Ser Gly Thr Asn Lys Arg Asp Lys Gln 85 90 95 Tyr GlnAla His Arg Ala Phe Gly Asp Arg Arg Asn Gly Val Ile Ser 100 105 110 AlaArg Thr Tyr Phe Tyr Ala Asn Glu Ala Lys Cys Asp Ser His Met 115 120 125Glu Thr Phe Leu Arg Cys Ile Glu Ala Ser Gly Arg Val Ser Asp Asp 130 135140 Gly Phe Ile Ala Ile Lys Leu Thr Ala Leu Gly Arg Pro Gln Phe Leu 145150 155 160 Leu Gln Phe Ser Glu Val Leu Ala Lys Trp Arg Cys Phe Phe HisGln 165 170 175 Met Ala Val Glu Gln Gly Gln Ala Gly Leu Ala Ala Met AspThr Lys 180 185 190 Leu Glu Val Ala Val Leu Gln Glu Ser Val Ala Lys LeuGly Ile Ala 195 200 205 Ser Arg Ala Glu Ile Glu Asp Trp Phe Thr Ala GluThr Leu Gly Val 210 215 220 Ser Gly Thr Met Asp Leu Leu Asp Trp Ser SerLeu Ile Asp Ser Arg 225 230 235 240 Thr Lys Leu Ser Lys His Leu Val ValPro Asn Ala Gln Thr Gly Gln 245 250 255 Leu Glu Pro Leu Leu Ser Arg PheThr Glu Glu Glu Glu Leu Gln Met 260 265 270 Thr Arg Met Leu Gln Arg MetAsp Val Leu Ala Lys Lys Ala Thr Glu 275 280 285 Met Gly Val Arg Leu MetVal Asp Ala Glu Gln Thr Tyr Phe Gln Pro 290 295 300 Ala Ile Ser Arg LeuThr Leu Glu Met Gln Arg Lys Phe Asn Val Glu 305 310 315 320 Lys Pro LeuIle Phe Asn Thr Tyr Gln Cys Tyr Leu Lys Asp Ala Tyr 325 330 335 Asp AsnVal Thr Leu Asp Val Glu Leu Ala Arg Arg Glu Gly Trp Cys 340 345 350 PheGly Ala Lys Leu Val Arg Gly Ala Tyr Leu Ala Gln Glu Arg Ala 355 360 365Arg Ala Ala Glu Ile Gly Tyr Glu Asp Pro Ile Asn Pro Thr Tyr Glu 370 375380 Ala Thr Asn Ala Met Tyr His Arg Cys Leu Asp Tyr Val Leu Glu Glu 385390 395 400 Leu Lys His Asn Ala Lys Ala Lys Val Met Val Ala Ser His AsnGlu 405 410 415 Asp Thr Val Arg Phe Ala Leu Arg Arg Met Glu Glu Leu GlyLeu His 420 425 430 Pro Ala Asp His Gln Val Tyr Phe Gly Gln Leu Leu GlyMet Cys Asp 435 440 445 Gln Ile Ser Phe Pro Leu Gly Gln Ala Gly Tyr ProVal Tyr Lys Tyr 450 455 460 Val Pro Tyr Gly Pro Val Met Glu Val Leu ProTyr Leu Ser Arg Arg 465 470 475 480 Ala Leu Glu Asn Ser Ser Leu Met LysGly Thr His Arg Glu Arg Gln 485 490 495 Leu Leu Trp Leu Glu Leu Leu ArgArg Leu Arg Thr Gly Asn Leu Phe 500 505 510 His Arg Pro Ala 515 3 1494DNA Mus musculus 3 atgttcgaga gattgatgaa gatgaccttc tatggccattttgtggctgg cgaagaccag 60 gagtctatca ggcctctgat ccggcacaac aaagcctttggtgttggctt tatcctggac 120 tatggagtgg aggaagatct gagccctgag gaggcggagcgcaaagagat ggagtcatgc 180 acttctgaag cagagagaga tggcagtgga gcaaataagagggagaagca gtatcaggtg 240 caccccgcct ttggagaccg cagagatggt gtcatcagtgcccgcaccta cttctatgcc 300 aatgaagcca agtgtgacaa ctacatggag aacttactgcagtgcatcaa ggcctcaggt 360 ggagccagtg atggtggttt ctcagccatt aagctcactgcactggggag accacagttt 420 ctgctgcagt tctcagacgt gctgaccagg tggagacggttcttccatca aatggctgca 480 gagcagggac aggctgggcg tgctgctgta gacacaaagctggaggtggc ggtgctccag 540 gacagcatcg caaagatggg catcgcatcc agggctgagattgaagggtg gttcacgcca 600 gagacgctgg gagtgtctgg caccgtggac ttgctggactggaacagcct cattgacagc 660 aggacccggc tctccaggca cttggtggtc cccaatgtgcagactggcca gctggagccc 720 ctgctgtcac ggttcactga ggaggaagag cagcagatgaaaaggatgct gcagaggatg 780 gatgtactgg ccaagaaagc aaaagaagca ggtgtgcgcctgatgattga tgctgagcag 840 agctacttcc aaccagccat cagccgcctg accctggagatgcagcgcag gttcaatgtg 900 gataagccgt tcatcttcaa cacattccag tgctacctcaaggatgccta tgacaatgtg 960 accttggata tggaactggc tcgccgtgag ggctggtgttccggggccaa gctggtacgt 1020 cgtgcataca tggcccaaga gcgtgtcagg gcagcagagatcggttatga agaccccatc 1080 aaccctacat atgaagccac caatgctatg taccacaggtgccttaacta tgttctggag 1140 gagctgaagc acagcaccaa ggcagaagtg atggtggcttcccacaacga ggacaccgtg 1200 cacttcackt tgtgcaggat gaaggagata ggcctgcatcctgctgatgg tcaggtgtgc 1260 ttcggacagc tgctggggat gtgtgaccaa atcagcttcccactaggcca ggcaggcttt 1320 cctgtgtaca agtatgtgcc ctatggccct gtgatggaggtactccctta cctgtcccgc 1380 cgtgccctgg agaacagcag catcatgaag ggtgctcagcgagagaggca gctgctatgg 1440 caggagctcc gcaggcggct gcgcactggc agcctcttccaccatccggc ctag 1494 4 497 PRT Mus musculus 4 Met Phe Glu Arg Leu MetLys Met Thr Phe Tyr Gly His Phe Val Ala 1 5 10 15 Gly Glu Asp Gln GluSer Ile Arg Pro Leu Ile Arg His Asn Lys Ala 20 25 30 Phe Gly Val Gly PheIle Leu Asp Tyr Gly Val Glu Glu Asp Leu Ser 35 40 45 Pro Glu Glu Ala GluArg Lys Glu Met Glu Ser Cys Thr Ser Glu Ala 50 55 60 Glu Arg Asp Gly SerGly Ala Asn Lys Arg Glu Lys Gln Tyr Gln Val 65 70 75 80 His Pro Ala PheGly Asp Arg Arg Asp Gly Val Ile Ser Ala Arg Thr 85 90 95 Tyr Phe Tyr AlaAsn Glu Ala Lys Cys Asp Asn Tyr Met Glu Asn Leu 100 105 110 Leu Gln CysIle Lys Ala Ser Gly Gly Ala Ser Asp Gly Gly Phe Ser 115 120 125 Ala IleLys Leu Thr Ala Leu Gly Arg Pro Gln Phe Leu Leu Gln Phe 130 135 140 SerAsp Val Leu Thr Arg Trp Arg Arg Phe Phe His Gln Met Ala Ala 145 150 155160 Glu Gln Gly Gln Ala Gly Arg Ala Ala Val Asp Thr Lys Leu Glu Val 165170 175 Ala Val Leu Gln Asp Ser Ile Ala Lys Met Gly Ile Ala Ser Arg Ala180 185 190 Glu Ile Glu Gly Trp Phe Thr Pro Glu Thr Leu Gly Val Ser GlyThr 195 200 205 Val Asp Leu Leu Asp Trp Asn Ser Leu Ile Asp Ser Arg ThrArg Leu 210 215 220 Ser Arg His Leu Val Val Pro Asn Val Gln Thr Gly GlnLeu Glu Pro 225 230 235 240 Leu Leu Ser Arg Phe Thr Glu Glu Glu Glu GlnGln Met Lys Arg Met 245 250 255 Leu Gln Arg Met Asp Val Leu Ala Lys LysAla Lys Glu Ala Gly Val 260 265 270 Arg Leu Met Ile Asp Ala Glu Gln SerTyr Phe Gln Pro Ala Ile Ser 275 280 285 Arg Leu Thr Leu Glu Met Gln ArgArg Phe Asn Val Asp Lys Pro Phe 290 295 300 Ile Phe Asn Thr Phe Gln CysTyr Leu Lys Asp Ala Tyr Asp Asn Val 305 310 315 320 Thr Leu Asp Met GluLeu Ala Arg Arg Glu Gly Trp Cys Ser Gly Ala 325 330 335 Lys Leu Val ArgArg Ala Tyr Met Ala Gln Glu Arg Val Arg Ala Ala 340 345 350 Glu Ile GlyTyr Glu Asp Pro Ile Asn Pro Thr Tyr Glu Ala Thr Asn 355 360 365 Ala MetTyr His Arg Cys Leu Asn Tyr Val Leu Glu Glu Leu Lys His 370 375 380 SerThr Lys Ala Glu Val Met Val Ala Ser His Asn Glu Asp Thr Val 385 390 395400 His Phe Thr Leu Cys Arg Met Lys Glu Ile Gly Leu His Pro Ala Asp 405410 415 Gly Gln Val Cys Phe Gly Gln Leu Leu Gly Met Cys Asp Gln Ile Ser420 425 430 Phe Pro Leu Gly Gln Ala Gly Phe Pro Val Tyr Lys Tyr Val ProTyr 435 440 445 Gly Pro Val Met Glu Val Leu Pro Tyr Leu Ser Arg Arg AlaLeu Glu 450 455 460 Asn Ser Ser Ile Met Lys Gly Ala Gln Arg Glu Arg GlnLeu Leu Trp 465 470 475 480 Gln Glu Leu Arg Arg Arg Leu Arg Thr Gly SerLeu Phe His His Pro 485 490 495 Ala 5 20 DNA Artificial SequenceDescription of Artificial SequencePRIMER 5 gaccaaatca gcttcccact 20 6 20DNA Artificial Sequence 6 cccttcatga tgctgctgtt 20 7 2240 DNA Musmusculus 7 agcgcgtctt cttgctgcgg tcggtggcac cacgcgtcgc tgccctctcaaccaaaccgc 60 aagcccagga acagcctccc gcgagccctg aggctcttcg gggatgtggggcggccaagg 120 ctgtgcggcc gcctgtgcca gccgtggact tcaccaacac gcaggaggcgtatcgcagcc 180 ggcggagttg ggagttggtg cgcaacctgc tagtgctgcg gctgtgtgcgtcgccggtgc 240 tgctagcgca ccacgagcag ttgttccaag ttgccaggaa gcttctggggcaaaggatgt 300 tcgagagatt gatgaagatg accttctatg gccattttgt ggctggcgaagaccaggagt 360 ctatcaggcc tctgatccgg cacaacaaag cctttggtgt tggctttatcctggactatg 420 gagtggagga agatctgagc cctgaggagg cggagcgcaa agagatggagtcatgcactt 480 ctgaagcaga gagagatggc agtggagcaa ataagaggga gaagcagtatcaggtgcacc 540 ccgcctttgg agaccgcaga gatggtgtca tcagtgcccg cacctacttctatgccaatg 600 aagccaagtg tgacaactac atggagaact tactgcagtg catcaaggcctcaggtggag 660 ccagtgatgg tggtttctca gccattaagc tcactgcact ggggagaccacagtttctgc 720 tgcagttctc agacgtgctg accaggtgga gacggttctt ccatcaaatggctgcagagc 780 agggacaggc tgggcgtgct gctgtagaca caaagctgga ggtggcggtgctccaggaca 840 gcatcgcaaa gatgggcatc gcatccaggg ctgagattga agggtggttcacgccagaga 900 cgctgggagt gtctggcacc gtggacttgc tggactggaa cagcctcattgacagcagga 960 cccggctctc caggcacttg gtggtcccca atgtgcagac tggccagctggagcccctgc 1020 tgtcacggtt cactgaggag gaagagcagc agatgaaaag gatgctgcagaggatggatg 1080 tactggccaa gaaagcaaaa gaagcaggtg tgcgcctgat gattgatgctgagcagagct 1140 acttccaacc agccatcagc cgcctgaccc tggagatgca gcgcaggttcaatgtggata 1200 agccgttcat cttcaacaca ttccagtgct acctcaagga tgcctatgacaatgtgacct 1260 tggatatgga actggctcgc cgtgagggct ggtgttccgg ggccaagctggtacgtcgtg 1320 catacatggc ccaagagcgt gtcagggcag cagagatcgg ttatgaagaccccatcaacc 1380 ctacatatga agccaccaat gctatgtacc acaggtgcct taactatgttctggaggagc 1440 tgaagcacag caccaaggca gaagtgatgg tggcttccca caacgaggacaccgtgcact 1500 tcackttgtg caggatgaag gagataggcc tgcatcctgc tgatggtcaggtgtgcttcg 1560 gacagctgct ggggatgtgt gaccaaatca gcttcccact aggccaggcaggctttcctg 1620 tgtacaagta tgtgccctat ggccctgtga tgtaggtact cccttacctgtcccgccgtg 1680 ccctggagaa cagcagcatc atgaagggtg ctcagcgaga gaggcagctgctatggcagg 1740 agctccgcag gcggctgcgc actggcagcc tcttccacca tccggcctagtcaccgcagg 1800 agccttgccc acccgctcgt actccactca accccttacc tctggggcttcaggcggggc 1860 acagcttggg attgggctgg ggttccttaa cccaacctgc ccagacacagttcacctttt 1920 tatgcccaag gctttttatg cccaaggcgg gatttcatca gtggacagttcctgaggaac 1980 agtgcccaag atggtcgtct ggtcacagag gctgccttct gggacttcctgtaccccaag 2040 gaacagacac tcaggagtgg ggtcagttag agcccctggg agctgccccactaatttgag 2100 taagcactga ccacttctgc aggttacaga gccctagtcc aggattaaccttctgccagg 2160 gtctaaccca ttttccctgc actgggcaga ggacagacta ggaagcctgtttagtcaata 2220 aatcatcctg taacagagtc 2240 8 452 PRT Mus musculus 8 MetPhe Glu Arg Leu Met Lys Met Thr Phe Tyr Gly His Phe Val Ala 1 5 10 15Gly Glu Asp Gln Glu Ser Ile Arg Pro Leu Ile Arg His Asn Lys Ala 20 25 30Phe Gly Val Gly Phe Ile Leu Asp Tyr Gly Val Glu Glu Asp Leu Ser 35 40 45Pro Glu Glu Ala Glu Arg Lys Glu Met Glu Ser Cys Thr Ser Glu Ala 50 55 60Glu Arg Asp Gly Ser Gly Ala Asn Lys Arg Glu Lys Gln Tyr Gln Val 65 70 7580 His Pro Ala Phe Gly Asp Arg Arg Asp Gly Val Ile Ser Ala Arg Thr 85 9095 Tyr Phe Tyr Ala Asn Glu Ala Lys Cys Asp Asn Tyr Met Glu Asn Leu 100105 110 Leu Gln Cys Ile Lys Ala Ser Gly Gly Ala Ser Asp Gly Gly Phe Ser115 120 125 Ala Ile Lys Leu Thr Ala Leu Gly Arg Pro Gln Phe Leu Leu GlnPhe 130 135 140 Ser Asp Val Leu Thr Arg Trp Arg Arg Phe Phe His Gln MetAla Ala 145 150 155 160 Glu Gln Gly Gln Ala Gly Arg Ala Ala Val Asp ThrLys Leu Glu Val 165 170 175 Ala Val Leu Gln Asp Ser Ile Ala Lys Met GlyIle Ala Ser Arg Ala 180 185 190 Glu Ile Glu Gly Trp Phe Thr Pro Glu ThrLeu Gly Val Ser Gly Thr 195 200 205 Val Asp Leu Leu Asp Trp Asn Ser LeuIle Asp Ser Arg Thr Arg Leu 210 215 220 Ser Arg His Leu Val Val Pro AsnVal Gln Thr Gly Gln Leu Glu Pro 225 230 235 240 Leu Leu Ser Arg Phe ThrGlu Glu Glu Glu Gln Gln Met Lys Arg Met 245 250 255 Leu Gln Arg Met AspVal Leu Ala Lys Lys Ala Lys Glu Ala Gly Val 260 265 270 Arg Leu Met IleAsp Ala Glu Gln Ser Tyr Phe Gln Pro Ala Ile Ser 275 280 285 Arg Leu ThrLeu Glu Met Gln Arg Arg Phe Asn Val Asp Lys Pro Phe 290 295 300 Ile PheAsn Thr Phe Gln Cys Tyr Leu Lys Asp Ala Tyr Asp Asn Val 305 310 315 320Thr Leu Asp Met Glu Leu Ala Arg Arg Glu Gly Trp Cys Ser Gly Ala 325 330335 Lys Leu Val Arg Arg Ala Tyr Met Ala Gln Glu Arg Val Arg Ala Ala 340345 350 Glu Ile Gly Tyr Glu Asp Pro Ile Asn Pro Thr Tyr Glu Ala Thr Asn355 360 365 Ala Met Tyr His Arg Cys Leu Asn Tyr Val Leu Glu Glu Leu LysHis 370 375 380 Ser Thr Lys Ala Glu Val Met Val Ala Ser His Asn Glu AspThr Val 385 390 395 400 His Phe Thr Leu Cys Arg Met Lys Glu Ile Gly LeuHis Pro Ala Asp 405 410 415 Gly Gln Val Cys Phe Gly Gln Leu Leu Gly MetCys Asp Gln Ile Ser 420 425 430 Phe Pro Leu Gly Gln Ala Gly Phe Pro ValTyr Lys Tyr Val Pro Tyr 435 440 445 Gly Pro Val Met 450 9 2389 DNA Homosapiens 9 taatgagagg gaaaacaagt atgaagctgt gtggctgaaa ccgtcttggcaagttaggga 60 aagaaaacgg aagtcactgg ggctgatcac agtgctaagc atgagagcactgcaagatga 120 ggtcacggag gtgggcaggg accggcttgt gccaggcctt gctggcagggtgaagagttt 180 gccttttctc tgcgtacaat ggaaaggaga agaggtttta agcaagagaatggcttggtc 240 atgtgtatgt ctttgagaca ccctggctag tctatgtatg atgcaaaaggtgggtggggc 300 agggtgacaa gaaaatactg ttccggagct tcctgtggct gtgcctataagaggtggtgg 360 tggtggtgtg gaaggaggtg tggcagtgaa taaacagaga tgtagaaacagcgtgtacat 420 atattttaag gaacactgag gacgtgatgc tggaatttgt gatgagagagtggaaaaaat 480 ccaggaaact tctaggacag aggctattca acaagctcat gaagatgaccttctatgggc 540 attttgtagc cggggaggac caggagtcca tccagcccct gcttcggcactacagggcct 600 tcggtgtcag cgccatcctg gactatggag tggaggagga cctgagccccgaggaggcag 660 agcacaagga gatggagtcc tgcacctcag ctgcggagag ggatggcagtggcacgaata 720 agcgggacaa gcaataccag gcccaccggg ccttcgggga ccgcaggaatggtgtcatca 780 gtgcccgcac ctacttctac gccaatgagg ccaagtgcga cagccacatggagacattct 840 tgcgctgcat cgaagcctca ggtagagtca gcgatgacgg cttcatagccattaagctca 900 cagcactggg gagaccccag tttctgctgc agttctcaga ggtgctggccaagtggaggt 960 gcttctttca ccaaatggct gtggagcaag ggcaggcggg cctggctgccatggacacca 1020 agctggaggt ggcggtgctg caggaaagtg tcgcaaagtt gggcatcgcatccagggctg 1080 agattgagga ctggttcacg gcagagaccc tgggagtgtc tggcaccatggacctgctgg 1140 actggagcag cctcatcgac agcaggacca agctgtccaa gcacctggtagtccccaacg 1200 cacagacagg acagctggag cccctgctgt cccggttcac tgaggaggaggagctacaga 1260 tgaccaggat gctacagcgg atggatgtcc tggccaagaa agccacagagatgggcgtgc 1320 ggctgatggt ggatgccgag cagacctact tccagccggc catcagccgcctgacgctgg 1380 agatgcagcg gaagttcaat gtggagaagc cgctcatctt caacacataccagtgctacc 1440 tcaaggatgc ctatgacaat gtgaccctgg acgtggagct ggctcgccgtgagggctggt 1500 gttttggggc caagctggtg cggggcgcat acctggccca ggagcgagcccgtgcggcag 1560 agatcggcta tgaggacccc atcaacccca cgtacgaggc caccaacgccatgtaccaca 1620 ggtgcctgga ctacgtgttg gaggagctga agcacaacgc caaggccaaggtgatggtgg 1680 cctcccacaa tgaggacaca gtgcgcttcg cactgcgcag gatggaggagctgggcctgc 1740 atcctgctga ccaccaggtg tactttggac agctgctagg catgtgtgaccagatcagct 1800 tcccgctggg ccaggccggc taccccgtgt acaagtacgt gccctatggccccgtgatgg 1860 aggtgctgcc ctacttgtcc cgccgtgccc tggagaacag cagcctcatgaagggcaccc 1920 atcgggagcg gcagttgctg tggctggagc tcttgaggcg gctccgaactggcaacctct 1980 tccatcgccc tgcctagcac ccgccagcac accctcagcc tccagcaccccccgcccccg 2040 cccaggccat caccacagct gcagccaacc ccatcctcac acagattcaccttttttcac 2100 cccacacttg cagagctgct ggaggtgagg tcaggtgcct cccagccctgcccagagtat 2160 gggcactcag gtgtgggccg aacctgatac ctgcctggga cagccactggaaacttttgg 2220 gaactctcct cgaatgtgtg gcccaaggcc cccacctctg tgacccccatgtccttggac 2280 ctagaggatt gtccaccttc tgccaaggcc agcccacaca gcccgagccccttggggagc 2340 agtggccggg ctggggaggc ctgcctggtc aataaaccac tgttcctgc2389

What is claimed is:
 1. An isolated variant allele of a human prolinedehydrogenase (PRODH) gene, wherein said PRODH gene comprises a DNAsequence of SEQ ID NO:1, and said variant allele comprises a DNAsequence having at least one variation in SEQ ID NO:1 wherein the atleast one variation comprises: a G to A transition in the third positionof codon 83; a C to T transition in the first position of codon 101; a Gto A transition in the second position of codon 101; a C to T transitionin the first position of codon 247; a C to T transition in the thirdposition of codon 342; a C to T transition in the third position ofcodon 421; an A to G transition in the second position of codon 437; a Tto C transition in the first position of codon 497; or a combinationthereof.
 2. An isolated nucleic acid molecule hybridizable to anisolated variant allele of a human PRODH gene of claim 1 under standardhybridization conditions.
 3. The isolated variant allele of claim 1,detectably labeled.
 4. The isolated nucleic acid molecule of claim 2,detectably labeled.
 5. The isolated variant allele of claim 3 whereinsaid detectable label comprises a radioactive element, a chemical whichfluoresces, or an enzyme.
 6. The isolated nucleic acid molecule of claim4, wherein said detectable label comprises a radioactive element, achemical which fluoresces, or an enzyme.
 7. An isolated variant alleleof a human PRODH gene, which encodes a variant proline dehydrogenasecomprising an amino acid sequence comprising at least one variation inSEQ ID NO:2, wherein said at least one variation comprises: Arg101Trp;Arg101Glu; Glu437Arg; or a combination thereof.
 8. An isolated varianthuman proline dehydrogenase comprising an amino acid sequence comprisingat least one variation in SEQ ID NO:2, wherein said at least onevariation comprises: Arg101Trp; Arg101Glu; Glu437Arg; or a combinationthereof.
 9. An antibody having the variant proline dehydrogenase ofclaim 8 as an immunogen.
 10. The antibody of claim 9, which is apolyclonal antibody.
 11. The antibody of claim 9, which is a monoclonalantibody.
 12. The antibody of claim 9, which is a chimeric antibody. 13.The antibody of claim 9, detectably labeled.
 14. The antibody of claim13, wherein said detectable label comprises a radioactive element, achemical which fluoresces, or an enzyme.
 15. A cloning vector comprisingan origin of replication and an isolated variant allele of a human PRODHgene, wherein said human PRODH gene comprises a DNA sequence of SEQ IDNO:1, and said variant allele comprises a DNA sequence having at leastone variation in SEQ ID NO:1, wherein said at least one variationcomprises: a G to A transition in the third position of codon 83; a C toT transition in the first position of codon 101; a G to A transition inthe second position of codon 101; a C to T transition in the firstposition of codon 247; a C to T transition in the third position ofcodon 342; a C to T transition in the third position of codon 421; an Ato G transition in the second position of codon 437; a T to C transitionin the first position of codon 497; or a combination thereof.
 16. Acloning vector comprising an origin of replication and an isolatednucleic acid molecule hybridizable under standard hybridizationconditions to an isolated variant allele of a PRODH gene, wherein saidPRODH gene comprises a DNA sequence of SEQ ID NO:1, and said isolatedvariant allele comprises a DNA sequence having at least one variation inSEQ ID NO:1, wherein said at least one variation comprises: a G to Atransition in the third position of codon 83; a C to T transition in thefirst position of codon 101; a G to A transition in the second positionof codon 101; a C to T transition in the first position of codon 247; aC to T transition in the third position of codon 342; a C to Ttransition in the third position of codon 421; an A to G transition inthe second position of codon 437; a T to C transition in the firstposition of codon 497; or a combination thereof.
 17. The cloning vectorof either of claim 15 or 16, wherein said cloning vector is selectedfrom the group consisting of E. coli, bacteriophages, plasmids, and pUCplasmid derivatives.
 18. The cloning vector of claim 17, whereinbacteriophages further comprise lambda derivatives, plasmids furthercomprise pBR322 derivatives, and pUC plasmid derivatives furthercomprise pGEX vectors, or pmal-c, pFLAG.
 19. An expression vectorcomprising an isolated variant allele of a human PRODH gene operativelyassociated with a promoter, wherein said human PRODH gene comprises aDNA sequence of SEQ ID NO:1, and said isolated variant allele comprisesa DNA sequence comprising at least one variation in SEQ ID NO:1, whereinsaid at least one variation and a variant allele of the presentinvention comprises a DNA sequence having at least one variation in SEQID NO:1 wherein the at least one variation comprises: a G to Atransition in the third position of codon 83; a C to T transition in thefirst position of codon 101; a G to A transition in the second positionof codon 101; a C to T transition in the first position of codon 247; aC to T transition in the third position of codon 342; a C to Ttransition in the third position of codon 421; an A to G transition inthe second position of codon 437; a T to C transition in the firstposition of codon 497; or a combination thereof.
 20. An expressionvector comprising an isolated nucleic acid molecule operativelyassociated with a promoter, wherein the isolated nucleic acid moleculeis hybridizable under standard hybridization conditions to an isolatedvariant allele of a human PRODH gene, wherein said human PRODH comprisesa DNA sequence of SEQ ID NO:1, and said variant allele comprises a DNAsequence having at least one variation in SEQ ID NO:1, wherein said atleast one variation comprises: a G to A transition in the third positionof codon 83; a C to T transition in the first position of codon 101; a Gto A transition in the second position of codon 101; a C to T transitionin the first position of codon 247; a C to T transition in the thirdposition of codon 342; a C to T transition in the third position ofcodon 421; an A to G transition in the second position of codon 437; a Tto C transition in the first position of codon 497; or a combinationthereof.
 21. The expression vector of either of claim 19 or 20, whereinsaid promoter is selected from the group consisting of the immediateearly promoters of hCMV, early promoters of SV40, early promoters ofadenovirus, early promoters of vaccinia, early promoters of polyoma,late promoters of SV40, late promoters of adenovirus, late promoters ofvaccinia, late promoters of polyoma, the lac the trp system, the TACsystem, the TRC system, the major operator and promoter regions of phagelambda, control regions of fd coat protein, 3-phosphoglycerate kinasepromoter, acid phosphatase promoter, and promoters of yeast α matingfactor.
 22. A unicellular host transformed or transfected with anexpression vector comprising an isolated variant allele of a human PRODHgene operatively associated with a promoter, wherein a human PRODH genecomprises a DNA sequence of SEQ ID NO:1, and said variant allelecomprises a DNA sequence having at least one variation in SEQ ID NO:1,wherein said at least one variation comprises: a G to A transition inthe third position of codon 83; a C to T transition in the firstposition of codon 101; a G to A transition in the second position ofcodon 101; a C to T transition in the first position of codon 247; a Cto T transition in the third position of codon 342; a C to T transitionin the third position of codon 421; an A to G transition in the secondposition of codon 437; a T to C transition in the first position ofcodon 497; or a combination thereof.
 23. A unicellular host transformedwith an expression vector comprising an isolated nucleic acid moleculeoperatively associated with a promoter, wherein the isolated nucleicacid molecule is hybridizable under standard hybridization conditions toan isolated variant allele of a human PRODH gene, wherein said humanPRODH gene comprises a DNA sequence of SEQ ID NO:1, and said variantallele comprises a DNA sequence having at least one variation in SEQ IDNO:1, wherein said at least one variation comprises: a G to A transitionin the third position of codon 83; a C to T transition in the firstposition of codon 101; a G to A transition in the second position ofcodon 101; a C to T transition in the first position of codon 247; a Cto T transition in the third position of codon 342; a C to T transitionin the third position of codon 421; an A to G transition in the secondposition of codon 437; a T to C transition in the first position ofcodon 497; or a combination thereof.
 24. The unicellular host of eitherof claim 22 or 23, wherein said host is selected from the groupconsisting of E. coli, Pseudonomas, Bacillus, Strepomyces, yeast, CHO,R1.1, B-W, L-M, COS1, COS7, BSC1, BSC40, BMT10 and Sf9 cells.
 25. Amethod of producing an a variant human PRODH protein comprising an aminoacid sequence comprising at least one variation in SEQ ID NO:1, whereinsaid at least one variation comprises: Arg101Trp; Arg101Glu; Glu437Arg;or a combination thereof, wherein said method comprising the steps of:(a) culturing a unicellular host of either of claim 22 or 23 underconditions that provide for expression of said variant human PRODHprotein; and (b) recovering said variant human PRODH protein from saidunicellular host, said culture, or both.
 26. A method for detecting asusceptibility to, or the presence of, schizophrenia or a disease ordisorder related thereto, wherein the method comprises measurement ofthe levels of activity of an enzyme in a bodily sample, wherein saidenzyme is involved in proline catabolism, and comparison of said levelsto a standard, whereby modulated levels indicate the susceptibility to,or the presence of, schizophrenia or a disease or disorder relatedthereto.
 27. The method for detecting a susceptibility of claim 26,wherein a disease or disorder related to schizophrenia comprisesobsessive compulsive disorder (OCD), bipolar disorder (BP) and majordepressive disorder (MDD).
 28. The method for detecting a susceptibilityas set forth in claim 26, wherein said enzyme is proline dehydrogenase(PRODH) comprising an amino acid sequence of SEQ ID NO:2, and reducedlevels of PRODH in a bodily sample as compared to a standard indicatesthe susceptibility to, or the presence of schizophrenia or a disease ordisorder related thereto.
 29. A method for determining a susceptibilityin a subject to schizophrenia, or a disease or disorder related thereto,wherein the method comprises the steps of: (a) removing a bodily samplefrom the subject, wherein the sample comprises a PRODH gene; (b)determining whether the PRODH gene of the bodily sample comprises a DNAsequence comprising a variation in SEQ ID NO:1 comprising a T to Ctransition in the first position of codon 497, such that the presence ofsaid at least one variation in said PRODH gene is indicative of thesubject's susceptibility to schizophrenia or a disease or disorderrelated thereto, relative to the susecptibility of a standard.
 30. Themethod of 29, wherein where the variation of PRODH gene is indicative ofan increased susceptibility of the subject to schizophrenia or a diseaseor disorder related thereto relative to the susceptibility of astandard.
 31. An assay system for screening drugs and other agents forability to treat schizophrenia or a disease or disorder related thereto,wherein the assay system comprises: (a) culturing an observable cellulartest colony inoculated with a drug or agent; (b) harvesting a cellularextract from said cellular test colony; and (c) examining said extractfor the presence of PRODH; wherein an increase or a decrease in a levelof activity of said PRODH in said test colony compared to level ofactivity of PRODH in the control indicates the ability of the drug tomodulate the production, stability, degradation or activity of saidPRODH.
 32. The assay for screening drugs and other agents of claim 31,wherein an increase in the level or activity of said PRODH in said testcolony compared to a control is indicative of the potential of the drugto treat schizophrenia or a disease or disorder related thereto.
 33. Atest kit to facilitate diagnosis and treatment of schizophrenia or adisease or disorder related thereto, comprising: (a) a predeterminedamount of a detectably labeled specific binding partner of a PRODH; (b)other reagents; and (c) directions for use of said kit.
 34. The test kitof claim 33, wherein said labeled immunochemically reactive component isselected from the group consisting of polyclonal antibodies to thePRODH, monoclonal antibodies to the PRODH, fragments thereof, andmixtures thereof.
 35. A test kit to facilitate diagnosis and treatmentof schizophrenia or a disease or disorder related thereto in aeukaryotic cellular sample, comprising: (a) PCR oligonucleotide primerssuitable PRODH detection; (b) other reagents; and (c) directions for useof said kit.
 36. A test kit to facilitate diagnosis and treatment ofschizophrenia or a disease or disorder related thereto in a subject,wherein the test kit comprises: (a) PCR oligonucleotide primers suitablefor detection of an isolated variant allele of a PRODH gene in a bodilysample of the subject comprising a DNA sequence having a variation inSEQ ID NO:1 comprising a T to C transition in the first position ofcodon 497; (b) other reagents; and (c) directions for using said kit.37. A method of treating schizophrenia or a disease or disorder relatedthereto, comprising administering to a mammal a therapeuticallyeffective amount of a PRODH comprising an amino acid sequence of SEQ IDNO:2, a conservative variant thereof, a fragment thereof, or an analogor derivative thereof.
 38. The method of claim 37 wherein said PRODHcomprising an amino acid sequence of SEQ ID NO:2 is administered tomodulate the course of therapy where said PRODH is being co-administeredwith one or more additional therapeutic agents.
 39. A method ofdetermining the schizophrenic-related pharmacological activity of acompound comprising: administering the compound to a mammal; determiningthe level of activity of PRODH comprising an amino acid sequence of SEQID NO:2 in the mammal; and comparing the level of activity of PRODH inthe mammal to the level of activity of PRODH in a standard to which thecompound was not administered, wherein an increase in the level ofactivity of PRODH in the mammal relative the level of activity of PRODHin the standard is indicative of the ability of the compound to treatschizophrenia or a disease or disorder related thereto.
 40. An isolatednucleic acid molecule comprising the DNA sequence of SEQ ID NO:1,degenerate variants thereof, fragments thereof, or analogs orderivatives thereof.
 41. The isolated nucleic acid molecule of claim 40detectably labeled.
 42. The isolated nucleic acid molecule of claim 41,wherein the detectable label comprises a radioactive element, a chemicalwhich fluoresces, or an enzyme.
 43. An isolated nucleic acid moleculehybridizable under standard hybridization conditions to an isolatednucleic acid molecule comprising a DNA sequence of SEQ ID NO:1,degenerate variants thereof, fragments thereof, or analogs orderivatives thereof.
 44. The isolated nucleic acid molecule of claim 43,detectably labeled.
 45. The isolated nucleic acid molecule of claim 49,wherein the detectable label comprises a radioactive element, a chemicalwhich fluoresces, or an enzyme.
 46. An isolated nucleic acid moleculewhich encodes human proline dehydrogenase protein, wherein the proteincomprises an amino acid sequence of SEQ ID NO:2.
 47. The isolatednucleic acid molecule of claim 46, comprising a DNA sequence of SEQ IDNO:1.
 48. An isolated protein comprising an amino acid sequence of SEQID NO:2, conservative variants thereof, fragments thereof, or analogs orderivatives thereof.
 49. An isolated nucleic acid molecule comprisingthe DNA sequence of SEQ ID NO:3, degenerate variants thereof, fragmentsthereof, or analogs or derivatives thereof.
 50. The isolated nucleicacid molecule of claim 49 detectably labeled.
 51. The isolated nucleicacid molecule of claim 50, wherein the detectable label comprises aradioactive element, a chemical which fluoresces, or an enzyme.
 52. Anisolated nucleic acid molecule hybridizable under standard hybridizationconditions to an isolated nucleic acid molecule comprising a DNAsequence of SEQ ID NO:3, degenerate variants thereof, fragments thereof,or analogs or derivatives thereof.
 53. The isolated nucleic acidmolecule of claim 52, detectably labeled.
 54. The isolated nucleic acidmolecule of claim 53, wherein the detectable label comprises aradioactive element, a chemical which fluoresces, or an enzyme.
 55. Anisolated nucleic acid molecule which encodes human proline dehydrogenaseprotein, wherein the protein comprises an amino acid sequence of SEQ IDNO:4.
 56. The isolated nucleic acid molecule of claim 55, comprising aDNA sequence of SEQ ID NO:3.
 57. An isolated protein comprising an aminoacid sequence of SEQ ID NO:4, conservative variants thereof, fragmentsthereof, or analogs or derivatives thereof.
 58. A method of identifyingdrugs or agents useful in treating schizophrenia or a disease,comprising the steps of: performing an first pre-pulse inhibition test(PPI) test on an F3 generation mouse from a cross Pro/Re X C57B1/6Jwild-type, wherein the F3 generation mouse has two copies within itsgenome of an isolated variant allele of a Prodh gene comprising a DNAsequence of SEQ ID NO:7 which are capable of expressing a mutant Prodhcomprising an amino acid sequence of SEQ ID NO:8, to obtain a firstpercentage of inhibition of startle response; administering thepotential drug or agent to the F3 generation mouse from a cross ofPro/Re X C57B1/6J wild-type; performing a second PPI test on the F3generation mouse from a cross of Pro/Re X C57B1/6J wild-type to obtain asecond percentage of inhibition of startle response; and comparing thefirst percentage to the inhibition of startle response with the secondpercentage of startle response, wherein an increase in percentage ofinhibition in the second percentage of inhibition relative to the firstpercentage of inhibition is indicative of the ability of the drug oragent to treat schizophrenia or a disease or disorder related thereto.59. A method of identifying drugs or agents useful in treatingschizophrenia or a disease, comprising the steps of: a) administeringthe drug or agent to an F3 generation mouse from a cross of Pro/Re XC57B1/6J wild-type, wherein the F3 generation mouse has two copieswithin its genome of an isolated variant allele of a Prodh genecomprising a DNA sequence of SEQ ID NO:7 which are capable of expressinga mutant Prodh comprising an amino acid sequence of SEQ ID NO:8; b)performing a PPI test on the F3 generation mouse from a cross of Pro/ReX C57B1/6J wild-type administered the drug or agent to obtain apercentage of inhibition of the startle response in the mouse; and c)comparing the percentage of inhibition of the startle response in F3generation mouse from a cross of Pro/Re X C57B1/6J wild-typeadministered the drug with the percentage of inhibition of the startleresponse in an F3 generation mouse from a cross of Pro/Re X C57B1/6Jwild-type, wherein the F3 generation mouse has two copies within itsgenome of an isolated Prodh gene comprising a DNA sequence of SEQ IDNO:3 which are capable of expressing a Prodh comprising an amino acidsequence of SEQ ID NO:4, such that the percentage of inhibition of thestartle response in the medicated mouse is statistically equivalent tothe percentage of inhibition in the mouse capable of expressing Prodhcomprising a DNA sequence of SEQ ID NO:4, then the drug or agent has theability to treat schizophrenia or a disease or disorder related thereto.60. A method for identifying a drug or agent for use in treatingschizophrenia or a disease or disorder related thereto, comprising thesteps of: a) administering the drug or agent to an F3 generation mousefrom a cross of Pro/Re X C57B1/6J wild-type, wherein the F3 generationmouse has two copies within its genome of an isolated variant allele ofa Prodh gene comprising a DNA sequence of SEQ ID NO:7 which are capableof expressing a mutant Prodh comprising an amino acid sequence of SEQ IDNO:8; b) performing a PPI test on the F3 generation mouse from a crossof Pro/Re X C57B1/6J wild-type to obtain a percentage of inhibition ofthe startle response in the F3 generation mouse from a cross of Pro/Re XC57B1/6J wild-type which was administered the drug or agent; and c)comparing the percentage of inhibition of the startle response in the F3generation mouse from a cross of Pro/Re X C57B1/6J wild-type with thepercentage of inhibition of the startle response in an unmedicated F3generation mouse from a cross of Pro/Re X C57B1/6J wild-type, whereinthe F3 generation mouse has two copies within its genome of an isolatedvariant allele of a Prodh gene comprising a DNA sequence of SEQ ID NO:7which are capable of expressing a mutant Prodh comprising an amino acidsequence of SEQ ID NO:8, wherein an increase in percentage of inhibitionin the percentage of inhibition in the medicated F3 generation mouserelative to the percentage of inhibition in the unmedicated F3generation mouse is indicative of the ability of the drug or agent totreat schizophrenia or a disease or disorder related thereto.